Enhancement of Inducible Nitric Oxide Synthase ... - ACS Publications

Subscriber access provided by NEW YORK UNIV

Article

Enhancement of Inducible Nitric Oxide Synthase Activity by Low Molecular Weight Peptides Derived from Protamine: A Potential Therapy for Chronic Rhinosinusitis Anant S. Balijepalli, Adam T. Comstock, Xuewei Wang, Gary C. Jensen, Marc B. Hershenson, Mark A. Zacharek, Umadevi S. Sajjan, and Mark E. Meyerhoff Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00110 • Publication Date (Web): 15 May 2015 Downloaded from http://pubs.acs.org on May 19, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Enhancement of Inducible Nitric Oxide Synthase Activity by Low Molecular Weight Peptides Derived from Protamine: A Potential Therapy for Chronic Rhinosinusitis Anant S. Balijepalli,†,‡ Adam T. Comstock,§ Xuewei Wang,† Gary C. Jensen, † Marc B. Hershenson,§ Mark A. Zacharek,⊥ Umadevi S. Sajjan,§ and Mark E. Meyerhoff†,* †Department of Chemistry, University of Michigan, 930 N. University, Ann Arbor, MI, 48109, United States ‡Current address: Department of Biomedical Engineering, Boston University, 44 Cummington, Boston, MA, 02215, United States §Department of Pediatrics and Communicable Diseases, University of Michigan Health System, 1150 W. Medical Center, Ann Arbor, MI, 48109, United States

⊥Department of Otolaryngology, University of Michigan Health System, 1500 E. Medical

Center, Ann Arbor, MI, 48109, United States

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 38

ABSTRACT: Nitric oxide (NO) is a key immune defense agent that is produced from L-arginine in the airways by leukocytes and airway epithelial cells, primarily via inducible nitric oxide synthase (iNOS). Deficiencies in nasal NO levels have been associated with diseases such as primary ciliary dyskinesia and chronic rhinosinusitis.

Herein, we demonstrate a proof-of-

concept regarding a potential new therapeutic approach for such disorders. We show that arginine-rich low molecular weight peptides derived from the FDA-approved protamine (obtained from salmon sperm) (LMWPs) are effective at significantly raising NO production in both RAW 264.7 mouse macrophage and LA4 mouse epithelial cell lines. LMWP is produced using a stable, easily produced immobilized thermolysin gel column followed by size-exclusion purification.

Monomeric L-arginine induces concentration-dependent increases in NO

production in stimulated RAW 264.7 and LA4 cells, as measured by stable nitrite in the cell media. In stimulated RAW 264.7 cells, LMWP significantly increases nitric oxide synthase (iNOS) expression and total NO production 12 to 24 h post-treatment compared to cells given equivalent levels of monomeric L-arginine. For stimulated LA4 cells, LMWPs are effective in significantly increasing NO production compared to equivalent L-arginine monomer concentrations over 24 h, but do not substantially enhance iNOS expression. The use of the arginase inhibitor S-boronoethyl-L-cysteine (BEC) in combination with LMWPs results in even higher NO production by stimulated RAW 264.7 cells and LA4 cells. Increases in NO due to LMWPs, compared to L-arginine, occur only after 4 h, which may be due to iNOS elevation rather than increased substrate availability.

KEYWORDS: nitric oxide, protamine, chronic rhinosinusitis, s-boronoethyl-l-cysteine, cellpenetrating peptides, iNOS, arginase, cystic fibrosis


Page 3 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC/Abstract Graphic: LMWP

NO(g)

LMWP

iNOS

L-Arg

NO(g)

L-Arginine

Bacteria exo-Arg iNOS



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 38

INTRODUCTION Nitric oxide (NO), produced by an isoform of the enzyme inducible nitric oxide synthase (iNOS), is central to the mammalian immune response and has been reported to be an important antimicrobial agent used by macrophages to kill L. major, M. bovis, and M. tuberculosis1,2,3 among numerous other microbes. Similarly, adequate NO production by iNOS within sinonasal epithelial cells helps prevent upper airway infection, especially sinus infections.4 Deficiencies in NO production can lead to a dysfunctional immune response, biofilm formation, or in some cases, septic shock.5 These deficiencies can either be genetic3 or induced by pathogens that target pathways regulating NO production.6 One such target is L-arginine, which is the substrate for all isoforms of NOS and is a major metabolic mediator within the urea cycle. Intracellular levels of L-arginine in airway cells lacking a complete urea cycle, such as bronchial epithelial cells, endothelial cells, and alveolar macrophages, are tightly regulated by the enzyme arginase, which is constitutively expressed in two isoforms, arginase I and II.7,8,9,10 In hepatocytes, the regulation of iNOS activity at physiological L-arginine levels suggests a close link to the urea cycle.7 Arginase has been shown to be co-induced following stimulation in a delayed manner,11 hinting at an intrinsic switch by which macrophages can attenuate their own immune response and prevent oxidative and nitrosative damage.

Many bacterial species induce arginase I

expression in murine macrophages,6,12,13,14 thereby skewing the macrophage phenotype to promote their survival.

In addition, several species of bacteria such as B. subtilis,15 B.

anthracis,16 and H.pylori17 are also known to express their own arginases, and in B. subtilis,18 B. anthracis,19 and S. aureus,18 functionally independent NOS, which may further limit the availability of L-arginine for iNOS activity. Increases in arginase expression may reduce NO production by iNOS and cause respiratory immune dysfunction, leading to the persistence of 4 ACS Paragon Plus Environment

Page 5 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


bacteria in the form of biofilms, as observed in patients with chronic rhinosinusitis and cystic fibrosis.20,21 A therapeutic approach that allows for the augmentation of iNOS activity and/or reduction of arginase activity would counteract these deficiencies.

Endogenous NO production can be stimulated in the respiratory tract by nebulizing Larginine,22,23 but this approach may not be efficacious, particularly in the presence of chronic bacterial infections. This is because L-arginine uptake by membrane transporters is slow,24 and increased host and bacterial arginase expression, as well as NOS expression, may readily metabolize L-arginine, thus reducing the extracellular and intracellular pools of L-arginine available for NOS activity. In contrast, it has been shown that in a variety of cell types,25,26 full delivery of low molecular weight protamine via active endocytic mechanisms,25 which are not possessed by most bacteria, can occur as quickly as 30 minutes.25 and in endothelial cells increase NO production from the blood vessel walls.27 In addition, the use of an arginase inhibitor has been shown to increase NO production and decrease bacterial burden in mice infected with Salmonella.28 Herein, we describe a modified method to prepare low molecular weight arginine-rich peptides (LMWPs) from protamine, an FDA-approved drug. We further examine the effects of the LMWP preparation compared to the L-arginine monomer in combination with an arginase inhibitor, s-boronoethyl-L-cysteine (BEC), on NO production in murine macrophages and airway epithelial cells.

EXPERIMENTAL SECTION Protamine

(Grade

X,

from

salmon

sperm),

thermolysin

(from

Bacillus

thermoproteolyticus Rokko), lipopolysaccharide from E. Coli, Sephadex G-15 (medium), N-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 38

hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, Bradford Reagent, Larginine free base, L-lysine free-base, SILAC RPMI 1640, SILAC Ham’s 12 media, and mouse anti-β-actin antibodies were purchased from Sigma-Aldrich (St. Louis, MO).

Murine

recombinant interferon (IFN)-γ, human recombinant interleukin (IL)-1β, and murine recombinant tumor necrosis factor (TNF)-α were purchased from Peprotech (Rocky Hill, NJ). BEC, and polyclonal rabbit anti-mouse iNOS antibody were purchased from Cayman Chemical (Ann Arbor, MI).

Laemmli buffer, goat anti-mouse and goat anti-rabbit IgG horseradish

peroxidase-conjugated IgG antibodies were obtained from Bio-Rad (Hercules, CA). SuperSignal West Pico chemiluminescence substrate was obtained from Thermo Scientific (Rockford, IL). RAW 264.7 and LA4 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). RPMI 1640 culture media, Cell-Dissociation Buffer, Ham’s F12 media, and fetal bovine serum (FBS) were obtained from Life Technologies (Grand Island, NY).

Preparation of LMWPs from Protamine. Protamine (10 mg) was dissolved at 70 oC in 1 ml of 10 mM Tris-HCl (pH 8.0) buffer containing 20 mM NaCl and 10 mM CaCl2. The solution was then passed via a peristaltic pump into a thermolysin-bound silica column (see results section) submerged in a 70 oC water bath. Thermolysin conjugation efficiency (expressed as a mean ± SEM of three conjugation experiments) was measured by passing 100 µL of filtrate collected from the conjugation reaction through a sterile 0.2 µm syringe filter (Millipore, Billerica, MA) and adding to 900 µL of Bradford reagent. After 20 min of incubation, the absorbance at 595 nm, measured on a Perkin-Elmer 25 UV/VIS Spectrophotometer (Waltham, MA), was compared to a calibration curve prepared using a wide concentration range of thermolysin solutions. The total calculated mass of thermolysin in the conjugation filtrate was


Page 7 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


compared to the total weighed mass of thermolysin to generate a w/w conjugation percentage. The protamine solution was cycled through this enzyme column for 3 h to ensure complete digestion and the column was completely dehydrated by filtration through a sterile 0.2 µm syringe filter. The collected filtrate was also analyzed by the Bradford assay for digestion efficiency by adding 100 µL of digestion effluent to 900 µL of Bradford reagent and incubating for 20 min before measuring absorbance at 595 nm. Digestion efficiency was calculated by dividing the absorbance of the effluent at 595 nm by the absorbance at 595 nm of 100 µL of a 10 mg/mL of protamine mixed with 900 µL of Bradford reagent.

Purification and Storage of LMWP. A 20 cm length Sephadex G-15 column was prepared (diameter = 1.5 cm) and the column was equilibrated by passing 3 column volumes of 10 mM Tris-HCl (pH 8.0) buffer through the column before sample loading at less than 30% of the column volume for peptide separation. Fractions of 0.5 mL were collected by gravity elution. Fractions were assayed for peptide content via absorbance at 210 nm by UV/VIS absorbance measurements and salt content by a conductivity measurement in order to generate a chromatogram.

The

initially

eluted

fractions

containing

the

target

fragments

VSRRRRRGGRRRR and VSRRRRRGGRRRRR ([M+H]+ = 1724.3 and 1880.5, respectively) were isolated, subjected to lyophilization, and stored at -20 oC.

Cell Culture. RAW 264.7 and LA4 cells were cultured in RPMI 1640 and Ham’s F12 medium, respectively. Each medium contained 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 100 µg/ml streptomycin. As iNOS, the primary contributor in host antimicrobial defense,1,2,3,4 is an inducible isoform, both RAW 264.7 and LA4 cells were stimulated by inflammatory cytokines in all experiments. For experiments involving the addition of L-arginine 7 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 38

monomer or LMWP, cells were cultured in SILAC RPMI 1640 or SILAC Ham’s F12 media partially constituted with L-arginine (Media 1: RPMI with 400 µM L-arginine, Media 2: Ham’s F12 with 400 µM L-arginine) and L-lysine (400 µM) and supplemented with 10% FBS (note: In the presence of FBS but no additional L-arginine, cells did not produce a detectable level of NO). Unless otherwise indicated, RAW 264.7 cells were plated at a density of 5 x 105 cells per well in 24 well plates, and LA4 cells were plated at a density of 2 x 105 cells per well in 6 well plates. After cell adhesion to the culture dishes, LA4 cells were stimulated with a mixture of mouse recombinant IFN-γ (10 ng/ml), human recombinant IL-1β (10 ng/ml) and mouse recombinant TNF-α (2 ng/ml). RAW 264.7 cells were stimulated with combination of LPS (20 ng/ml) and mouse recombinant IFN-γ (10 ng/ml). Cells were then treated with L-arginine (10 mM stock) or LMWP (1 mM stock) to a final concentration of 400 µM total L-arginine added. In some experiments, cells were treated with 90 µM BEC with the added LMWP/L-arginine. Cell culture supernatant was collected at indicated times and stored at 4o C for nitrite analysis as an indicator of NO production. Cell lysates were prepared and stored at -20 oC for Western blot analyses.

Nitrite Analysis. Media samples collected from cell culture experiments were analyzed for nitrite concentration using a Sievers Nitric Oxide Analyzer (NOA) (GE Analytical Instruments, Boulder, CO). Briefly, a reaction cell was prepared with a N2-purged solution (2 mL) of 0.5 M H2SO4 and 0.5 M KI in order to reduce nitrite in the sample to NO. A 0.05 mL sample of the test media was then added, and the generated NO was collected by a N2 sweep gas and delivered to the instrument for reaction with ozone to produce excited nitrogen dioxide. The release of photons from this intermediate is measured by a photomultiplier tube within the NOA to quantitate the gas phase NO levels in ppbv. The data was integrated over time in order to determine total nitrite content of the samples. NO generation was allowed to return to baseline 8 ACS Paragon Plus Environment

Page 9 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


before addition of successive samples.

Western Blot. Cells were washed 3x in cold phosphate buffered saline solution (pH 7.4) and lysed for 30 min on ice in 10 mM Tris buffer pH 8.0 containing 0.1% SDS, 1%Triton X-100, 0.1% sodium deoxycholate, and complete protease inhibitors (Roche, Indianapolis, IN). Cell lysates were centrifuged at 13,000 × g for 20 min at 4°C, and protein concentrations of the cleared cellular lysates were determined by the Lowry assay. The samples were mixed 3:1 with 4x Laemmli sample buffer, boiled for 5 min, and 30 µg aliquots were separated on an 8% SDSPAGE gel. The proteins were transferred to a 0.2 µm pore nitrocellulose membrane (Millipore), which was then blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 for 1 h at 25° C. The membrane was incubated with a 1:5,000 dilution of polyclonal rabbit anti-iNOS antibody or 1:10,000 monoclonal mouse anti-β-actin antibody overnight at 4°C and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG for 1 h at 25°C. The membrane was then incubated with the chemiluminescence substrate. Finally, blots were exposed on X-ray film for 30 s and developed using a Kodak XOmat Automatic Processor (Kodak, Rochester, NY). The images were scanned and the band intensities were quantified by Image J (NIH, Bethesda, MD) and expressed as fold change over β-actin.

RESULTS Preparation and Purification of Low Molecular Weight Protamine. Immobilization of thermolysin to cleave protamine has been previously proposed by David, et al. (2012)29 using amine-functionalized silica sol-gel microparticles, produced from sodium silicate 9 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 38

passed through an ion-exchange column. In these prior studies a glutaraldehyde cross-linker was employed to link the particles to the primary amines on the exterior of thermolysin.

We

modified this thermolysin immobilization procedure to use inexpensive and widely available fumed silica particles functionalized with 3-aminopropyltriethoxysilane followed by conjugation to a N-hydroxysuccinimidyl ester of thermolysin. A flow diagram of the process to generate these functionalized silica particles is shown in Figure 1B. We observed conjugation efficiencies of 92.5 ± 2.7 wt% of added enzyme to the silica substrate.

An example of the primary structure of protamine from salmon sperm is shown in Figure 1A, along with the peptides expected after protamine is in contact with immobilized thermolysin. Interestingly, the Bradford reagent displays very little sensitivity to LMWP peptides as opposed to cell penetrating peptides containing aromatic residues (Figure S1) and even protamine itself. This important distinction allows for a quantitative measure of efficiency of digestion by comparing the color upon reaction with Bradford reagent of an unreacted 10 mg/mL solution of protamine to that of the collected enzyme filtrate. As shown in Figure 1C, the enzyme column is stable over multiple uses, and 3 h of substrate cycling through the column results in digestion efficiencies of 93.1% on the first use with an observed minimum digestion efficiency of at least 81.0% with subsequent repeated use of the column over a 14 d period.

To isolate the target LMWP fragments and exchange buffer composition to one with a low salt concentration, a Sephadex G-15 size-exclusion chromatography column was prepared as described in the Experimental Methods section. The molecular weights of TDSP4 and TDSP5 lie near 2 kDa, and as a result, should elute from the 1.5 kDa cutoff column in a low-salt buffer


Page 11 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Tris-HCl 10 mM, pH 8.0) immediately after the void volume and before the lower molecular weight constituents. The chromatogram (Figure S2) displays six clear peaks in order of molecular weight corresponding to partially digested protamine, TDSP5, TDSP4, TDSP3, TDSP2, and TDSP1. As expected, the conductivity of the column effluent rises as the lower molecular weight constituents are eluted from the column, corresponding to the higher salt content of the digestion buffer. The fractions containing LMWP were pooled and subjected to lyophilization and the resulting powder was weighed to determine yield. A mass spectrum obtained by MALDI-TOF of the purified LMWP is shown in Figure 1D, showing that the primary peptides isolated have [M+H]+ m/z values of 1880.5 and 1724.3 corresponding to TDSP5 and TDSP4, respectively.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 38

Figure 1. A: Primary structure of protamine, Grade X from salmon sperm and the smaller digest fragments as determined by amino acid analysis.30 B: Flow diagram of thermolysinsilica conjugation process. C: Stability of thermolysin-silica column over 14 d of re-use. D: Mass spectrum of LMWP obtained by enzyme column method.


Page 13 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Inducible Nitric Oxide Synthase Activity and Expression in RAW 264.7 and LA4 Cells is Dependent on Extracellular Arginine Levels. The L-arginine Km for iNOS has been determined to lie in the low µM range (~5 µM),31 yet previous literature has demonstrated that NO synthesis can increase significantly when RAW 264.7 cells are activated in media containing L-arginine concentrations from 10 µM to 1.6 mM.6 This dependence of NO synthesis on L-arginine concentration has been attributed to increased iNOS expression and implies that diminished L-arginine availability can significantly attenuate NO production. As shown in Figure 2A, RAW 264.7 cells co-stimulated with LPS and IFN-γ (cytomix 1) express iNOS 24 h after stimulation, while unstimulated cells do not express detectable levels of iNOS. The nitrite content in the media of unstimulated (0.54 ± 0.11 µM) and stimulated cells (56.99 ± 0.48 µM) 24 h post-stimulation, shown in Figure 2B, corroborates the analysis of iNOS expression. LA4 cells, when stimulated by a mixture of IFN-γ, IL-1β, and TNFα (cytomix 2), also express NOS2, while unstimulated cells display no detectable expression (Figure 2C). The nitrite content of the collected media (Figure 2D) in each case again confirms the activity of iNOS, as it increases from 0.27 ± 0.22 µM in unstimulated cells to 7.84 ± 0.19 µM in stimulated cells.

When plated in media of varying L-arginine concentrations, we found that, in accordance with prior literature,6 RAW 264.7 cells display L-arginine-dependent NO synthesis during a 24 h period over a range of 10 µM to 1100 µM L-arginine (Figures 2E and 2F). Nitrite levels increased from 0.42 ± 0.04 µM to 11.08 ± 0.59 µM at 100 µM L-arginine to 56.99 ± 0.48 µM at 1100 µM L-arginine. Similar L-arginine dependence was also observed in LA4 cells (Figures 2G and 2H). Nitrite levels were found to increase with increasing concentration of arginine, 13 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 38

exhibiting saturation at 400 µM. Indeed, total NO production appears to peak at near this Larginine level for LA4 cells, with nitrite levels reaching a maximum of roughly 9 µM. iNOS expression in LA4 cells was similarly dependent on extracellular L-arginine concentration. In fact, arginine-induced iNOS expression may protect against substrate limitation due to arginase induction31 by pro-inflammatory stimuli.


Page 15 of 38

D"9

B"70

A" CM1

+

-

***"

β-actin

C" CM2

+

-

7

50

6

40

5 4

30

3

20

2

10

iNOS NOS2

1 0

0 Control

β-actin

Unstimulated

CM1

Treatment

E" L-Arginine

1000 µM 400 µM 100 µM

CM2

Treatment

G" L-Arginine 1000 µM

NOS2 iNOS

NOS2 iNOS

β-actin F"70

β-actin

400 µM 100 µM 10 µM

H" 40

CM1

Fold increase over 10 µM arginine

Control

***"

8

NO2- concentration (µM)

iNOS NOS2


60

60


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50 40 30 20 10

35 30 25 20 15 10 5 0

0 5 µM

20 µM

100 µM

400 µM

1100 µM

10 µM

100 µM

400 µM

1000 µM

[L-arginine]

[L-arginine]



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 38

Figure 2. A: Representative immunoblot: Stimulation of RAW 264.7 cells with cytomix 1 (10 ng/mL IFNg and 20 ng/mL LPS) results in NOS2 expression after 24 h in media 1. B: Nitrite concentrations of media in stimulated RAW 264.7 cells are ~60 fold higher (unpaired, onetailed t-test p-value < 0.001) compared to unstimulated RAW 264.7 cells. Data is an average ± s.d. of four independent experiments. C: Representative immunoblot: Stimulation of LA4 cells with cytomix 2 (10 ng/mL IFNg, 20 ng/mL LPS, 10 ng/mL IL-1B, and 2 ng/mL TNFa) results in NOS2 expression after 24 h. D: Nitrite concentrations of media in stimulated LA4 cells are ~15 fold higher (unpaired one-tailed t-test p-value < 0.001) than unstimulated LA4 cells. Data is an average ± s.d. for three independent experiments each. E: Representative immunoblot: Expression of NOS2 in RAW 264.7 cells is dependent on media L-arginine concentration as expression is shown to increase as concentration are modulated from 100 – 1000 µM. F: Nitrite concentrations in the media of RAW 264.7 cells incubated in L-arginine concentrations from 10 – 1000 µM. Data is an average ± s.d. for six independent experiments per concentration. G: Representative immunoblot: Expression of NOS2 in LA4 cells is dependent on media L-arginine concentration as expression increases as L-arginine concentration increases from 10 – 1000 µM. H: Nitrite concentrations in media of LA4 cells as L-arginine concentrations are varied from 1-1100 µM. Data is an average ± s.d. for n=4 independent experiments per concentration.

Low Molecular Weight Protamine Treatment Increases NO Production in RAW 264.7 and LA4 Cells. Although iNOS expression is L-arginine dependent, Larginine transmembrane transport is slow and can be inhibited by other cationic amino acids such as lysine and ornithine. We hypothesized that both RAW 264.7 and LA4 cells would rapidly take up the larger, arginine-rich LMWP species (TDSP4 and TDSP5), subsequently enhancing iNOS expression and NO production compared to equivalent concentrations of monomeric Larginine.

As shown in Figure 3A, supplementation of stimulated RAW 264.7 cells in media 1 with 40 μM LMWP results in a significant increase in NO production over 20 h compared to cells in media 1 not given LMWP (1.28 ± 0.08 vs. 0.70 ± 0.14 NOS2/β-actin). LMWP itself did not


Page 17 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


cause induction of iNOS for unstimulated cells in media 1 (see methods section). In addition, we found that iNOS expression in stimulated RAW 264.7 cells in media 1 is significantly enhanced upon treatment with LMWP compared to stimulated cells in media 1 not exposed to LMWP (Figure S3). Addition of 40 µM LMWP to RAW 264.7 cells resulted in a significant increase in NO production compared to cells given additional 400 μM L-arginine over 20 h in cultures preincubated with 20 μM (39.52 ± 2.88 vs. 31.53 ± 1.10 μM), 100 μM (41.35 ± 2.04 vs. 32.18 ± 1.03 μM), and 400 μM L-arginine (46.88 ± 1.64 vs. 38.60 ± 1.14 μM) (Figure 3B). Since RAW 264.7 cells pre-cultured in low-arginine environments (as low as 20 μM) exhibit increased NO generation with LMWP treatment, it is clear that the presence of L-arginine is not necessary for the beneficial action of LMWP species.

In order to determine if LMWP more rapidly provides substrate for iNOS compared to Larginine, we stimulated RAW 264.7 cells in various arginine-deficient conditions (10, 100, and 400 μM L-arginine) prior to addition of either 40 µM LMWP or 400 µM L-arginine and measured media nitrite content at 1, 2, and 4 h after treatment. We found that treatment with LMWP or L-arginine led to nearly identical NO production over the first four hours (before significant changes to iNOS expression can occur) for each level of iNOS expression (Figure S4). For the three different incubation concentrations of L-arginine and over the first three hours, LMWP on average was only 1.01 ± 0.06 times more effective than L-arginine. This data, coupled with the low L-arginine Km for iNOS, implies that the beneficial effect of L-arginine or LMWP supplementation does not lie in increased substrate availability, but rather increased iNOS expression.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 38

Indeed, as shown in Figure 3C, a significant difference between NO synthesis by stimulated RAW 264.7 cells in media 1 as a result of L-arginine (5.91 ± 0.44 μM) and LMWP addition (8.52 ± 0.38 μM) can be observed as early as 12 h post-treatment. In addition, the difference in NO generated between L-arginine and LMWP supplementation continues to increase from 5.07 ± 0.75 μM after 16 h to 8.28 ± 2.00 μM after 24 h (Figure S5). These findings are mirrored in the gradually rising expression of iNOS in RAW 264.7 cells in media 1 (Figure 3D) as a result of 40 µM LMWP treatment that, after 26 h, is significantly higher (1.28 ± 0.08 NOS2/β-actin) than for cells given 400 µM L-arginine (0.88 ± 0.06 NOS2/β-actin) and control cells (0.70 ± 0.14 NOS2/β-actin).


Page 19 of 38

B" C"55

4.5 4 3.5

Concentration (µM)

400 µM L-arginine added 40 µM LMWP added

50


A"

45

3

***"

2.5

**" **"

*"

40 35 30

2

25 20

1.5

15

1

10

0.5

5

0

0 Control

LMWP

20 µM

Treatment

C" E"55 50

100 µM

400 µM

[L-arginine]

D"1.51.5 G" D"

12 hours 16 hours 24 hours

***"

1.4 1.4

40

1.2 1.2

Relative density Relative

45

1.3 1.3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Control Control

**" **"

L-Arginine L-Arginine LMWP

LMWP

1.1 1.1

35 30

11

0.9 0.9

25

0.8 0.8

20

***"

15

**"

10

0.7 0.7 0.6 0.6 0.5 0.5

5 0 Control

L-arginine

LMWP

0.4 0.4 10 10

12 12

14 14

Treatment

16 16

18 18

20 20

Time Time(hours) (hours)

22 22

24 24

26 26

28 28

Figure 3. A: Addition of 40 µM LMWP (400 µM total L-arginine) to stimulated RAW 264.7 cells (unfilled bars) results in increased NO production compared to control cells (black bars) (unpaired, two-tailed t-test p < 0.001) indicating successful entry into target cells and arginine availability. Four independent experiments were conducted per experimental condition (cell density of 5 x104 cells/well), and media was collected 20 h after stimulation. Average ± s.d. for n=4 experiments are shown. B: Treatment with LMWP is effective even for RAW 264.7 cells pre-incubated in low L-arginine environments (20, 100, 400 µM). Cells were incubated in these media for 6 h before stimulation and addition of either additional L-arginine or LMWP. Each experimental condition is comprised of four independent experiments with average ± s.d reported. Using unpaired, two-tailed t-test, *: p = 0.01, **: p = 0.002. C: Nitrite concentrations in the media of cells treated with no addition (control), L-arginine, and LMWP show that the LMWP is effective increasing NO production as compared to L-arginine from 12-24 h after stimulation and addition. Unpaired, two-tailed t-test, **: p = 0.0021, ***: p < 0.001. D: Densitometry of NOS2 immunoblot for cells treated with no addition (control), L-arginine, and LMWP over time shows LMWP effectively induces increasing NOS2 expression compared to Larginine up to 26 h after treatment (unpaired two-tailed t-test p = 0.0038). Each lane of blot was comprised of four independent experiments for a total n=12 for each treatment. Error bars are standard deviation of relative density for each protein band to assess for significant differences, comparatively. 19 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 38

Treatment with Low Molecular Weight Protamine and s-Boronoethyl-lcysteine synergistically increases NO Production in RAW 264.7 but not in LA4 cells. Prior studies have demonstrated the capacity of the competitive arginase inhibitor BEC to enhance iNOS activity in H. pylori-treated RAW 264.7 cells.6 It is important to note that arginase can be present in the extracellular environment during infection or chronic inflammation due to its production by pathogens15,16,17 or leukocytes.32 We hypothesize that BEC acts to protect delivered intracellular L-arginine and enhances NO production by both increasing iNOS expression and increasing substrate availability. Indeed, we found that addition of BEC (over a range of 26 μM to 155 μM) to stimulated RAW 264.7 cells in media 1 yielded increasing NO production, with an optimum concentration of 100 μM BEC (Figure S6). When treated with LMWP and BEC, RAW 264.7 cells in media 1 produced more NO (1.55 ± 0.03 fold increase over control cells) than the additive effect of LMWP and BEC alone (1.42 ± 0.04 fold increase over control cells) over a 24 h period (Figure 4A). There was no significant increase in NO production by cells treated with L-arginine and BEC. Treatment of stimulated LA4 cells in media 2 with LMWP and BEC resulted in elevation of NO compared to treatment with LMWP or BEC alone, but the effect was weaker than in RAW 264.7 cells (Figure 4B). Interestingly, the total NO production from stimulated RAW 264.7 cells shown in Figure 4A was not mirrored in the expression of iNOS over the same 24 h period (Figure 4C). We found that the expression of iNOS for stimulated RAW 264.7 cells treated with either LMWP/BEC or L-arginine/BEC increased to peaks of 1.12 ± 0.13 and 0.78 ± 0.13 NOS2/β-actin 16 h after treatment, but decreased after 26 h to 1.00 ± 0.18 and 0.58 ± 0.09, respectively. Treatment with LMWP, Larginine, or BEC alone led to continuously rising expression (Figure 4D). This data suggests that the large amount of NO produced from stimulated RAW 264.7 cells may have acted as 20 ACS Paragon Plus Environment

Page 21 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


negative feedback to repress iNOS expression. Similarly, the expression of iNOS in stimulated LA4 cells is diminished for treatment with LMWP and LMWP+BEC whose nitrite levels reached the ~9 µM limiting levels seen in Figure 2F (see Figure S8). We found that treatment of LA4 cells with BEC yielded a greater change in iNOS expression from control cells as opposed to RAW 264.7 cells (1.18 ± 0.04 vs. 1.09 ± 0.02 fold increase) (see Figure S8).



B"1.7 A"

C" B"

‡‡" ††" **"

1.6

1.4

Fold increase over control


1.5 1.4 1.3 1.2 1.1 1 0.9

†"

1.3

‡‡"

%"

**" 1.2

1.1

1

0.9 Stimulated

BEC

L-arginine

L-arginine and BEC

LMWP

LMWP and BEC

Control

BEC

L-Arginine

D" C" B"

BEC NOS2 iNOS β-actin

C" F"1.6 D" 1.6

- -

+

L-Arginine and BEC

LMWP

LMWP and BEC

Treatment

Treatment

- + -

+

Relative density density (iNOS/Actin) Relative (iNOS/Actin)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 38

L-Arginine L-Arginine L-Arginine andand BECBEC L-Arginine LMWP LMWP LMWP and BEC LMWP and BEC

1.4 1.4

**$$ **"

†" **" **,†$$

1.2 1.2 11

0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 00

10 10

12 12

14 14

16 16

18 18

20 20

22 22

24 24

26 26

28 28

Time (hours) Time (hours)

Figure 4. A: LMWP and BEC together synergistically increase NO production in RAW 264.7 cells (media 1) over 24 h (‡: paired, one-tailed t-test comparing treatment 6 (LMWP+BEC) to the added value of treatments 2 (BEC) and 5 (LMWP) p = 0.0072) and NO production is higher than either L-arginine+BEC or LMWP alone (†,*: paired, two-tailed t-test p-values = 0.0023,0.0026). B: LMWP is effective in increasing NO production in LA4 cells (media 2) over 24 h compared to control cells (*: paired, one-tailed t-test p-value = 0.0037) and those given an equivalent concentration of L-arginine (-: paired, one-tailed t-test p-value = 0.043). LMWP+BEC elicits a moderate increase in NO production and is statistically significant compared to treatment with L-arginine alone (‡: paired, one-tailed t-test p value = 0.0027) and BEC (†: paired, one-tailed t-test p value = .024). C: Representative immunoblots of NOS2 expression over 24 h in stimulated RAW 264.7 (media 1). Expression of NOS2 is higher for treatment with LMWP alone, but total NO production is higher for LMWP with BEC. D: Timedependent densitometry analysis of immunoblots of NOS2 in stimulated RAW 264.7 cells. LMWP+BEC peaks at 16 h (significant compared to L-arginine [*: paired, two-tailed t-test pvalue = 0.0089] and L-arginine+BEC [†: paired, two-tailed t-test p-value = 0.032) but falls after 26 h. Each point is comprised of four independent experiments. Error bars are standard deviation of relative density for each protein band to assess for significant differences, comparatively.


Page 23 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DISCUSSION The augmentation of endogenous NO production is an attractive therapeutic target, owing to NO’s role as a potent antimicrobial agent. Given the many millions of people who suffer from NO-related diseases such as chronic rhinosinusitis and ciliary dyskinesia, we chose to examine the effect of LMWP on augmentation of NO synthesis in murine macrophages and respiratory epithelial cells. LMWPs are derived from the FDA-approved protamine and prior research regarding LMWPs has mainly focused on its use as a potential antagonist/neutralizing agent for systemic heparin anticoagulation, as significant toxicity has been observed in this application with full-length protamine.33

Protamine obtained from salmon sperm is known to be

polydisperse and contains characteristic repeats of up to six arginine residue peptide segments (see Fig. 1A, above).

As described by Byun et al. (1999),34 the lower molecular weight

fragments of protamine containing these arginine repeats are capable of neutralizing heparin due to their high charge density at neutral pH, while exhibiting significantly reduced immunogenicity and toxicity compared to native protamine. Such protamine fragments have been purified and extensively characterized by Chang, et al. (2001)35 using amino acid analysis mass spectrometry, and five major constituents were identified and labeled as TDSP1 through TDSP5. Of these fragments, TDSP4 and TDSP5 present the greatest potential for cellular uptake while limiting immunogenicity and toxicity. These peptides are highly hygroscopic and completely insoluble in solvents such as methanol, ethanol, and isopropanol leading to numerous isolation and purification challenges. As a result, we designed an optimized, high-throughput, and low cost method for synthesis and purification of these peptides. In this work, the LMWPs were synthesized using an enzyme-conjugated column and



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 38

separated via simple size-exclusion chromatography on a Sephadex G-15 resin.

Digestion

efficiencies can be monitored by the Bradford assay, since the generated LMWPs exhibit very little binding affinity towards Coomassie Brilliant Blue G-250.

This may indicate that

stabilization of the sulfonic acid moiety of Coomassie Brilliant Blue G-250 requires the presence of hydrophobic residues in addition to basic residues. LMWP is highly hydrophilic due to the high number of arginine residues within their structures, and as a result, may not be able to interact with the hydrophobic core of Coomassie Brilliant Blue G-250. Therefore, to monitor the separation of the LMWPs on the Sephadex column, absorbance measurements at 210 nm were employed.

Our results demonstrate that the LMWP fraction isolated by our method is effective in increasing NO synthesis in two murine cell models - macrophages and airway epithelial cells that are relevant to respiratory disease in humans. The activity of iNOS is highly regulated by the presence of L-arginine in terms of both substrate availability and transcriptional regulation,36 and therefore, additional L-arginine supplementation can be efficacious in increasing iNOS expression and activity.

However, the presence of pathogens and/or chronic inflammatory

conditions can significantly attenuate the benefit of L-arginine supplementation due to the high levels of arginase and bacterial NOS in the environment.37 In contrast, the arginine-rich LMWP species, may not be substrates for arginase due to a lack of free α-amino and free α-carboxylate groups38 and have been shown to enter target cells rapidly through active endocytic processes,25,26,27 in contrast to slow24 and competitively-inhibited receptor-mediated L-arginine transport. It should be noted that the active site of iNOS is highly specific to L-arginine;39 therefore, an arginine-rich peptide itself likely cannot generate NO by iNOS. Indeed, even slight


Page 25 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


modifications to the L-arginine backbone40 and certain dipeptides40,41 containing both N- and Cterminal arginine lead to a large decrease in NO synthesis, compared to L-arginine monomer. Additionally, the tripeptide RRR was found to display greatly attenuated NO production in J774.2 cells, compared to monomeric L-arginine.42 The notion that protease activity within the cells is needed to increase NO production from iNOS in the presence of LMWP is supported by previous literature indicating that poly-arginines are known not to be good substrates for iNOS,40,41,42 In this work we demonstrate that additional NO synthesis immediately after addition of either L-arginine or LMWP is nearly identical. This implies that LMWP itself does not present a larger substrate reservoir for target cells than L-arginine. Yet, accumulated nitrite levels over 12, 16, and 24 h show a clear statistically significant advantage of LMWP over an equivalent total concentration of monomeric L-arginine, with the gap widening over time. Previous studies have shown that the clearance of the L-R7 peptide occurs over the course of 5 days in endothelial cells, but such clearance is inhibited by the use of D-R7.27 Further cell trafficking studies are required to determine if intracellular proteases such as trypsin-like proteases are responsible for increased iNOS activity due to LMWP.

We hypothesized that the arginase inhibitor BEC should bolster the efficacy of LMWP by further protecting intracellular and extracellular L-arginine from metabolism. We found moderate efficacy when using the inhibitor BEC alone and that arginase inhibition in the presence of LMWP causes a small but significant increase in NO compared to BEC alone in both LA4 and RAW 264.7 cells.

However, BEC alone was more effective in increasing NO

generation from LA4 rather than RAW 264.7 cells, possibly due to relatively higher arginase



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 38

expression in this cell type. Although the accumulated nitrite content in the media of RAW 246.7 cells treated with LMWP and BEC was greater than all other experimental treatments, the expression of iNOS in these cells after 24 h was lower than treatment with LMWP alone. This may be due to the fact that high NO can negatively regulate iNOS expression through feedback mechanisms. For instance, nitrosylation of the p65 subunit of NF-κB, which binds to activate iNOS transcription, is known to occur during high nitrosative stress.43

We propose that LA4 cells display an oxidative stress limit as evidenced by the limitation of NO biosynthesis at L-arginine concentrations exceeding 400 μM (with nitrite concentrations consistently peaking at ~9 μM). It is likely that a similar nitrosative-stress dependent mechanism underlies decreased iNOS expression in this cell type. The putative digestion of LMWP by intracellular proteases may in fact prolong the expression of iNOS by limiting oxidative stress. It is important to note that the production of reactive nitrogen and oxygen species is very tightly regulated, and even a 10-20% increase in NO production can have a significant antimicrobial effect.44 We anticipate that the expression of iNOS should continue to increase past the 26 h period observed in this study compared to control cells, further widening the gap in efficacy between LMWP and L-arginine. We will now seek to repeat these experiments using cultured human nasal epithelial cells, and then examine the effectiveness of this approach in an in vivo murine model of chronic rhinosinusitus. If results are promising, it is envisioned that a clinical formulation of LMWP alone or in combination with BEC within a nasal spray could be developed for easy administration to the sinuses of human subjects as a potential therapeutic in treating/preventing chronic rhinosinusitis.


Page 27 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Author Information Corresponding Author *Phone: 1-734-763-5916; e-mail: [email protected]. Fax: 734-647-4865. Notes The authors declare no competing financial interests.

Author Contributions Balijepalli and Comstock performed all experiments with the exception of Figure 1D (Wang) under the guidance of Drs. Sajjan, Meyerhoff, Hershenson and Zacharek. Dr. Jensen was involved in some of the very preliminary experiments that utilized synthetic polyarginine agents that became the basis for the efforts to use much less costly LMWP species to increase NO production by murine macrophages and epithelial cells.

Acknowledgements We would like to thank James Windak for assistance in performing MALDI-TOF Mass Spectrometry.

Supporting Information Available Data for the Bradford assay of LMWP, Sephadex G-15 separation of protamine digest, NOS2 expression with respect to LMWP and IFN-γ addition, NO production immediately following Larginine or LMWP addition, increasing difference in NO production over time between LMWP and L-arginine, NO production in response to increasing BEC concentration, and iNOS



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 38

expression in stimulated LA4 cells in response to L-arginine, BEC, and LMWP, is provided within the Supplemental Information file. This material is available free of charge via the Internet at http://pubs.acs.org.

REFERENCES

(1)

Vouldoukis, I.; Riveros-Moreno, V.; Dugas, B.; Ouaaz, F.; Bécherel, P.; Debré, P.; Moncada, S.; Mossalayi, M.D. The Killing of Leishmania major by Human Macrophages is Mediated by Nitric Oxide Induced After Ligation of the Fc Epsilon RII/CD23 Surface Antigen. Proc Natl Acad Sci U S A. 1995, 92, 7804-7808.

(2)

Nozaki, Y.; Hasegawa, Y.; Ichiyama, S.; Nakashima, I.; Shimokata, K. Mechanism of Nitric Oxide-Dependent Killing of Mycobacterium bovis BCG in Human Alveolar Macrophages. Infect Immun. 1997, 65, 3644-3647.

(3)

Nicholson, S.; Bonecini-Almeida Mda, G.; Lapa e Silva, J.R.; Nathan, C.; Xie, Q.W.; Mumford, R.; Weidner, J.R.; Calaycay, J.; Geng, J.; Boechat, N.; Linhares, C.; Rom, W. Ho, J.L. Inducible Nitric Oxide Synthase in Pulmonary Alveolar Macrophages from Patients with Tuberculosis. J Exp Med, 1996, 183, 2293-2302.

(4)

Lundberg, J.O. Nitric Oxide and the Paranasal Sinuses. Anat Rec (Hoboken). 2008, 291, 1479-1484.

(5)

Annane, D.; Sanquer, S.; Sébille, V.; Faye, A.; Djuranovic, D.; Raphael, J.C.; Gajdos, P.; Bellissant, E. Compartmentalised Inducible Nitric-Oxide Synthase Activity in Septic Shock. Lancet. 2000, 355, 1143-1148.


Page 29 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(6)

Chaturvedi, R.; Asim, M.; Lewis, N.D.; Algood, H.M.; Cover, T.L.; Kim, P.Y.; Wilson, K.T. L-arginine Availability Regulates Inducible Nitric Oxide Synthase-Dependent Host Defense Against Helicobacter pylori. Infect Immun. 2007, 75, 4305-4315.

(7)

Lerzynski, G.; Suschek, C.V.; Kolb-Bachofen, V. In Hepatocytes the Regulation of NOS2 Activity at Physiological L-arginine Levels Suggests a Close Link to the Urea Cycle. Nitric Oxide. 2006, 14, 300-308.

(8)

Klasen, S.; Hammermann, R.; Fuhrmann, M.; Lindemann, D.; Beck, K.F.; Pfeilschifter, J.; Racké, K. Glucocorticoids Inhibit Lipopolysaccharide-induced Up-regulation of Arginase in Rat Alveolar Macrophages. Br J Pharmacol. 2001, 132, 1349-1357.

(9)

Lindemann, D.; Racké, K. Glucocorticoid Inhibition of Interleukin-4 (IL-4) and Interleukin-13 (IL-13) Induced Up-regulation of Arginase in Rat Airway Fibroblasts. Naunyn Schmiedebergs Arch Pharmacol. 2003, 368, 546-550.

(10)

Que, L. G.; Kantrow, S.P.; Jenksinon, C.P.; Piantadosi, C.A.; Huang, Y.C. Induction of Arginase Isoforms in the Lung during Hyperoxia. Am J Physiol. 1998, 275, L96-L102.

(11)

Sharda, D.R.; Yu, S.; Ray, M.; Squadrito, M.L.; De Palma, M.; Wynn, T.A.; Morris Jr., S.M.; Hankey, P.A. Regulation of Macrophage Arginase Expression and Tumor Growth by the Ron Receptor Kinase. J Immunol. 2011, 187, 2181-2192.

(12)

Gobert, A.P.; Daulouede, S.; Lepoivre, M.; Boucher, J.L.; Bouteille, B.; Buguet, A.; Cespuglio, R.; Veyret, B. Vincendeau, P. L-arginine Availability Modulates Local Nitric Oxide Production and Parasite Killing in Experimental Trypanosomiasis. Infect Immun. 2000, 68, 4653-4657.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(13)

Page 30 of 38

Huang, J.; DeGraves, F.J.; Lenz, S.D.; Gao, D.; Feng, P.; Li, D.; Schlapp, T.; Kaltenboeck, B. The Quantity of Nitric Oxide Released by Macrophages Regulates Chlamydia-induced Disease. Proc Natl Acad Sci U S A. 2002, 99, 3914-3919.

(14)

Thurlow, L.R.; Hanke, M.L.; Fritz, T.; Angle, A.; Aldrich, A.; Williams, S.H.; Engebretsen, I.L.; Bayles, K.W.; Horswill, A.R.; Kielian, T. Staphylococcus aureus Biofilms Prevent Macrophage Phagocytosis and Attenuate Inflammation in vivo. J Immunol. 2011, 186, 6585-6596.

(15)

Yu, J.J.; Park, K.B.; Kim, S.G.; Oh, S.H. Expression, Purification, and Biochemical Properties of Arginase from Bacillus subtilis. J Microbiol. 2013, 51, 222-228.

(16)

Soru, E. Chemical and Immunological Properties of B. anthracis Arginase and its Metabolic Involvement. Mol Cell Biochem. 1983, 50, 173-183.

(17)

Zhang, X.; Zhang, J.; Zhang, R.; Guo, Y.; Wu, C.; Mao, X.; Guo, G.; Zhang, Y.; Li, D.; Zou, Q. Structural, Enzymatic, and Biochemical Studies on Helicobacter pylori Arginase. Int J Biochem Cell Biol. 2013, 45, 995-1002.

(18)

Gusarov, I.; Nudler, E. NO-mediated Cytoprotection: Instant Adaptation to Oxidative Stress in Bacteria. Proc Natl Acad Sci. 2005, 102, 13855-13860.

(19)

Shatalin, K.; Gusarov, I.; Avetissova, E.; Shatalina, Y.; McQuade, L.E.; Lippard, S.J.; Nudler, E. Bacillus antracis-derived Nitric Oxide is Essential for Pathogen Virulence and Survival in Macrophages. Proc Natl Acad Sci. 2007, 105, 1009-1013.

(20)

Foreman, A.; Psaltis, A.J.; Tan, L.W.; Wormald, P.J. Characterization of Bacterial and Fungal Biofilms in Chronic Rhinosinusitis. Am J Rhinol Allergy. 2009, 23, 556-561.


Page 31 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(21)

Singh, P.K.; Schaefer, A.L.; Parsek, M.R.; Moninger, T.O.; Welsh, M.J.; Greenberg, E.P. Quorum-sensing Signals Indicate that Cystic Fibrosis Lungs are Infected with Bacterial Biofilms. Nature. 2000, 407, 762-764.

(22)

Grasemann, H.; Kurtz, F.; Ratjen, F. Inhaled L-arginine Improves Exhaled Nitric Oxide and Pulmonary Function in Patients with Cystic Fibrosis. Am J Respir Crit Care Med. 2006, 174, 208-212.

(23)

Grasemann, H.; Tullis, E.; Ratjen, F. A Randomized Controlled Trial of Inhaled Larginine in Patients with Cystic Fibrosis. J Cyst Fibros. 2013, 12, 468-474.

(24)

Strobel, J.; Müller, F.; Zolk, O.; Endreß, B.; König, J.; Fromm, M.F.; Maas, R. Asymmetric Dimethylarginine (ADMA) by Cationic Amino Acid Transporter 2 (CAT2), Organic Cation Transporter 2 (OCT2) and Multidrug and Toxin Extrusion Protein 1 (MATE1). Amino Acids. 2013, 45, 989-1002.

(25)

Xia, H.; Gao, X.; Gu, G.; Liu, Z.; Zeng, N.; Hu, Q.; Song, Q.; Yao, L.; Pang, Z.; Jiang, X.; Chen, J.; Chen, H. Low Molecular Weight Protamine-functionalized Nanoparticles for Drug Delivery to the Brain after Intranasal Administration. Biomaterials. 2011, 32, 9888-9898.

(26)

Choi, Y.S.; Lee, J.Y.; Suh, J.S.; Kwon, Y.M.; Lee, S.J.; Chung, J.K.; Dong-Soo, L.; Yang, V.C.; Chung, C.P.; Park, Y.J. The Systemic Delivery of siRNAs by a Cell Penetrating Peptide, Low Molecular Weight Protamine. Biomaterials. 2009, 31, 14291443.

(27)

Uemura, S.; Fathman, C.G.; Rothbard, J.B.; Cooke, J.P. Rapid and Efficient Vascular Transport of Arginine Polymers Inhibits Myointimal Hyperplasia. Circulation. 2000, 102, 2629-2635.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(28)

Page 32 of 38

Lahiri, A.; Das, P.; Chakravortty, D. Arginase Modulates Salmonella induced Nitric Oxide Production in RAW264.7 Macrophages and is Required for Salmonella Pathogenesis in Mice Model of Infection. Microbes Infect. 2008, 10, 1166-1174.

(29)

David, A.E.; Gong, J.; Chertok, B.; Domszy, R.C.; Moon, C.; Park, Y.S.; Wang, N.S.; Yang, A.J.; Yang, V.C. Immobilized Thermolysin for Highly Efficient Production of Low-Molecular-Weight Protamine—An Attractive Cell-Penetrating Peptide for Macromolecular Drug Delivery Applications. J Biomed Mater Res A. 2012, 100, 211219.

(30)

Park, Y.J.; Chang, L.C.; Liang, J.F.; Moon, C.; Chung, C.P.; Yang, V.C. Nontoxic Membrane Translocation Peptide from Protamine, Low Molecular Weight Protamine (LMWP), for Enhanced Intracellular Protein Delivery: in vitro and in vivo Study. FASEB J. 2005, 19, 1555-1557.

(31)

Rao, K.M. Molecular Mechanisms Regulating iNOS Expression in Various Cell Types. J Toxicol Envrion Health B Crit Rev. 2000, 3, 27-58.

(32)

Rotonda, R.; Barisione, G.; Mastracci, L.; Grossi, F.; Orengo, A.M.; Costa, R.; Truini, M.; Fabbi, M; Ferrini, S.; Barbieri, O. IL-8 Induces Exocytosis of Arginase 1 by Neutrophil Polymorphonuclears in Nonsmall Cell Lung Cancer. Int J Cancer. 2009, 125, 887-893.

(33)

Chang, L.C.; Wrobleski, S.; Wakefield, T.W.; Lee, L.M.; Yang, V. C. Low Molecular Weight Protamine as Nontoxic Heparin/Low Molecular Weight Heparin Antidote (III): Preliminary in vivo Evaluation of Efficacy and Toxicity Using a Canine Model. AAPS PharmSci. 2001, 3, 24-31.


Page 33 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(34)

Byun, Y.; Singh, V.K.; Yang, V.C. Low Molecular Weight Protamine: A Potential Nontoxic Heparin Antagonist. Thromb Res. 1999, 94, 53-61.

(35)

Liang, J.F.; Zhen, L.; Chang, L.C.; Yang, V.C. A Less Toxic Heparin Antagonist—Low Molecular Weight Protamine. Biochemistry (Mosc.). 2003, 68 116-120.Kagemann, G.; Sies, H.; Schnorr, O. Limited Availability of L-arginine Increases DNA-binding Activity of NF-kappaB and Contributes to Regulation of iNOS Expression. J Mol Med (Berl). 2007, 85, 723-732.

(36)

Munder, M.; Schneider, H.; Luckner, C.; Giese, T.; Langhans, C.D.; Fuentes, J.M.; Kropf, P.; Mueller, I.; Kolb, A.; Modolell, M.; Ho, A.D. Suppression of T-cell Functions by Human Granulocyte Arginase. Blood. 2006, 108, 1627-1634.

(37)

Shishova, E.Y.; Costanzo, L.; Emig, F.A.; Ash, D.E.; Christianson, D.W. Probing the Specificity Determinants of Amino Acid Recognition by Arginase. Biochemistry. 2009, 48, 121-131.

(38)

Mori, M. Regulation of Nitric Oxide Synthesis and Apoptosis by Arginase and Arginine Recycling. J Nutr. 2007, 137, 1616S-1620S.

(39)

Iyengar, R.; Stuehr, D.J.; Marletta, M.A.; Macrophage Synthesis of Nitrite, Nitrate, and N-nitrosamines: Precursors and Role of the Respiratory Burst. Proc Natl Acad Sci. 1987, 84, 6369-6373.

(40)

Su, C.-L.; Austic, R.E.; The Utilization of Dipeptides Containing L-Arginine by Chicken Macrophages. Poul Sci. 1998, 77, 1852-1857.

(41)

Hecker, M.; Walsh, D.T.; Vane, J.R. On the Substrate Specificity of Nitric Oxide Synthase. FEBS. 1991, 294, 221-224.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(42)

Page 34 of 38

Kelleher, Z.T.; Matsumoto, A.; Stamler, J.S.; Marshall, H.E. NOS2 Regulation of NFkappaB by S-nitrosylation of p65. J Biol Chem. 2007, 282, 30667-30672.

(43)

El Kasmi, K.C.; Qualls, J.E.; Pesce, J.T.; Smith, A.M.; Thompson, R.W.; HenaoTamayo, M.; Basaraba, R.J.; König, T.; Schleicher, U.; Koo, M.S.; Kaplan, G.; Fitzgerald, K.A.; Tuomanen, E.I.; Orme, I.M.; Kanneganti, T.D.; Bogdan, C.; Wynn, T.A.; Murray, P.J. Toll-like Receptor-induced Arginase 1 in Macrophages Thwarts Effective Immunity Against Intracellular Pathogens. Nat Immunol. 2008, 9, 1399-1406.


B" Molecular Pharmaceutics

Page 35 of 38 A"

Protamine

PRRRRSSSRPVRRRRRPRVSRRRRRGGRRRRR

TDSP1 1

PRRRR

Dry

Introduce 0.5 wt% solution of APTES in anhydrous toluene

Incubate flask in N2 environment overnight

Dry

PRRRRSSSRP Wash 2x successively with toluene, ethanol, and water

RPVRRRRRPR

Dry particles at 120° C in vacuum oven for 1h

Incubate Thermolysin in 10 mM NHS and 40 mM EDC for 10 min

VSRRRRRGGRRRR VSRRRRRGGRRRRR Mix enzyme and APTES-silica for 30 min

D" F

Dry

Wash 2x successively Dry with ethanol and water

35000

Store functionalized particles at 4° C

TDSP5 (1880)

Intensity (a.u.)

30000

Percent digestion

2 TDSP2 3 4 TDSP3 5 6 TDSP4 7 TDSP5 8 9 10 11 12 C" D" 13120 14 15100 16 17 80 18 19 60 20 21 40 22 20 23 24 0 25 0 26 27

Generate silanol groups on surface of fumed silica particles

25000 20000 15000 10000

TDSP4 (1724)

5000

2

4

6

8

Day

10

ACS Paragon Plus Environment 0 12

14

16

700

1000

1300

1600

m/z

1900

2200

2500

A" CM1

-

+

Pharmaceutics B"Molecular 70

D"9

Page 36 of 38

***"


Fold increase over 10 µM arginine


C"


8 ***" 1 60 2 7 3 NOS2 iNOS 50 6 4 5 40 5 6 β-actin 7 4 30 8 3 9 20 10CM2 + 2 11 10 1 12 13NOS2 iNOS 0 0 14 Unstimulated CM2 Control CM1 15 Treatment Treatment β-actin 16 17 E" G" 18 400 µM 100 µM 1000 µM L-Arginine L-Arginine 1000 µM 400 µM 100 µM 10 µM 19 20 21NOS2 NOS2 iNOS iNOS 22 23 β-actin 24β-actin 25 F" H"40 2670 Control CM1 27 35 2860 2950 30 30 25 3140 32 20 3330 15 34 20 35 10 3610 5 37 38 0 0 10 µM 100 µM 400 µM 1000 µM 5 µM 20 µM 100 µM 400 µM 1100 µM 39 [L-arginine] [L-arginine] ACS Paragon Plus Environment 40 41 42

B" C"Molecular Pharmaceutics 55 400 µM L-arginine

4.5

A"of 38 Page 37 4

3

***"

2.5

40

**"

**"

*"

45


Concentration (µM)

3.5

35 30

2

25 20

1.5

15

1

10

0.5

5

0 Control

LMWP

0 20 µM

Treatment

100 µM

400 µM

[L-arginine]

G"1.51.5 D"

12 hours 16 hours 24 hours

***"


1.4 1.4 1.3 1.3 1.2 1.2

Relativedensity density Relative

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 C" E" 55 16 1750 1845 1940 2035 2130 2225 2320 24 15 25 2610 27 5 28 0 29 30

added 40 µM LMWP added

50

Control

Control

**" **"

L-Arginine

L-Arginine LMWP LMWP

1.1 1.1 11

0.9 0.9

***" **"

0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5

ACS Paragon Plus Environment 0.4 0.4 Control

L-arginine

Treatment

LMWP

10 10

12 12

14 14

16 16

18 18

20 20

Time Time(hours) (hours)

22 22

24 24

26 26

28 28

Molecular Pharmaceutics C"1.4 B" ‡‡" ††" **"

1 1.5 2 1.4 3 1.3 4 5 1.2 6 1.1 7 1 8 9 0.9 Stimulated 10 11 12 C"13 14 15 16 17 BEC 18 19 20 21NOS2 22 β-actin 23 24 25 26 27


1.6

BEC

L-arginine

L-arginine and BEC

LMWP

‡‡"

%"

**" 1.1

1

Control

BEC

L-Arginine

D" C" F"1.6 1.6

- -

+

L-Arginine and BEC

LMWP

LMWP and BEC

Treatment

Treatment

D" B"

iNOS

†"

1.2

0.9

LMWP and BEC

Page 38 of 38

1.3

- + -

+

Relative density density (iNOS/Actin) Relative (iNOS/Actin)


B"1.7 A"

L-Arginine L-Arginine L-Arginine andand BECBEC L-Arginine LMWP LMWP LMWP and BEC LMWP and BEC

1.4 1.4

**$$ **"

†" **" **,†$$

1.2 1.2 11

0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2

ACS Paragon Plus Environment 00 10 10

12 12

14 14

16 16

18 18

20 20

Time (hours) Time (hours)

22 22

24 24

26 26

28 28

Enhancement of Inducible Nitric Oxide Synthase ... - ACS Publications

Recommend Documents