Bioactivity of Peptide Analogs of the Neutrophil Chemoattractant, N-Acetyl-Proline-Glycine-Proline

Abstract

purpose. The release of N-acetyl-proline-glycine-proline (PGP), a chemoattractant resulting from direct alkaline hydrolysis of corneal proteins, is believed to be the initial trigger for neutrophil invasion into the alkali-injured cornea. The purpose of this study is twofold: (1) to compare the activity of N-acetyl-PGP with the bioactivities of other similar synthetic peptides in an effort to uncover information about this chemoattractant molecule, and (2) to test these peptide analogs as potential antagonists of N-acetyl-PGP.

methods. The polarization assay was used to measure the potential chemotactic response of human neutrophils to peptides. Bioactivity was expressed as the peptide concentration required to produce 50% neutrophil polarization (EC₅₀). Antagonist activity was expressed as the peptide concentration required to produce 50% inhibition (ID₅₀) of polarization activated by N-acetyl-PGP.

results. Peptide bioactivities (EC₅₀) were ranked as follows: APGPR (0.34 mM) > N-acetyl-PGP (0.5 mM) > N-(PGP)₄-PGLG (3 mM) = t-Boc-PGP (3 mM) > N-acetyl-PG (3.4 mM) > N-methyl-PGP (15 mM) = PGP (15 mM) > peptides without detectable activity (t-Boc-PGP-OMe, N-acetyl-P, PG, PGG, GP, GG and gly-pro-hyp). Peptides with no detectable bioactivity were tested as potential antagonists of neutrophil polarization induced by N-acetyl-PGP. Gly-Pro-Hyp inhibited N-acetyl-PGP activation of polarization at 20 mM (ID₅₀). No other synthetic peptide demonstrated a capacity for inhibition.

conclusions. The minimum requirement to elicit bioactivity was the presence of PGP alone or derivatives of PG in which the N-terminal proline is blocked. Using this approach, active and inactive mimetic peptides of N-acetyl-PGP were produced. The most active peptide, APGPR, was equal to or slightly greater than N-acetyl-PGP, suggesting that more potent analogs might be designed. Gly-pro-hyp was the only inactive peptide analog to inhibit the chemoattractant.

Alkali-injury of the eye provokes a severe inflammatory reaction, largely composed of neutrophils. ^{1 2} This acute inflammatory response is responsible for corneal ulcerations and perforations, so characteristic of this disease. The release of N-acetyl-proline-glycine-proline (PGP), a chemoattractant resulting from direct alkaline hydrolysis of corneal proteins, is believed to be the initial trigger for neutrophil invasion into the alkali-injured cornea. ^{3 4} This finding created an opportunity to determine the active portions of the N-acetyl-PGP molecule. One avenue of approach is to investigate the activity of peptide analogs, which are structurally related to N-acetyl-PGP. To develop these structure–activity relationships, we synthesized a series of peptide analogs and evaluated them in a system that measures neutrophil polarization, the earliest stage of chemotaxis. 

The major purpose of this study was to compare the activity of these peptides to the chemoattractant in an effort to uncover information about the relative importance of different portions of the N-acetyl-PGP molecule to biological activity. Additionally, these peptide analogs were tested as potential antagonists of N-acetyl-PGP. 

Materials and Methods

Materials

Hanks’ balanced salt solution (HBSS) was purchased from Gibco Laboratories (Chagrin Falls, OH). Calcium chloride, magnesium chloride, sodium chloride, glutaraldehyde, and Ficoll (type 400) were purchased from Sigma Chemical Co. (St. Louis, MO). Sodium phosphate monobasic and sodium phosphate dibasic were obtained from Fisher Scientific (Fair Lawn, NJ). Hypaque-76 was acquired from Winthrope Laboratories (New York, NY). Leukotriene B₄ (LTB₄) was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Solvents and reagents for peptide synthesis were purchased from VWR Scientific Products (West Chester, PA). Resin and suitably derivatized amino acids were purchased from Advanced Chem Tech (Louisville, KY) or PerSeptive Biosystem (Framingham, MA). 

Peptide Synthesis and Isolation

Several peptide analogs, N-acetyl-P, GG, PGG, gly-pro-hyp, and APGPR, were obtained from Sigma Chemical Co. Other peptides, PG, GP, PGP, N-acetyl-PG, t-Boc-PGP, and t-Boc-PGP-OMe, were synthesized by conventional solution phase peptide chemistry. However, for large-scale synthesis of N-acetyl-PGP, an alternative method was used to increase the yield of the product. In this method, the dipeptide t-Boc-PG was coupled to Pro-Merrifield resin (Nova Biochem, San Diego, CA) using the dicyclohexylcarbodiimide/1-hydroxybenzotriazole procedure. After the removal of the N-terminal protection and acetylation using acetic anhydride, the peptide was cleaved from the resin, using anhydrous hydrofluoric acid. The product was purified on a silica gel column, using chloroform:methanol (90:10, vol/vol) as the eluent. 

The starting compound for the synthesis of N-methyl-PGP was t-Boc-PGP. The removal of N terminus t-Boc and the addition of N-methyl group were accomplished by a modified Mannich reaction. ⁵ Briefly, the reaction mixture consisted of 30% formaldehyde, 98% formic acid (50 times molar excess to t-Boc-PGP), and freshly prepared palladium black. The reaction mixture was incubated overnight at 50°C, and the reaction was followed for completion by thin-layer chromatography. After the filtration of palladium black, the reaction mixture was diluted with water and lyophilized. 

The homogeneity of each peptide was confirmed by reversed-phase high-performance liquid chromatography (RP-HPLC) on a Vydac C18-analytical column equilibrated at a flow rate of 1.2 ml/min and eluted with a linear gradient from 0% to 30% acetonitrile in water (0.1% trifluoroacetic acid) in 30 minutes. Characterization was done using Electrospray Mass Spectrometry (Perkin-Elmer-Sciex API-3, Norwalk, CT). Quantitative amino acid analysis was performed to show the correct ratio of amino acids and to determine the peptide content for calculation of the final concentration. 

The PGP polymer, N-(PGP)₄-PGLG, was synthesized on a PerSeptive Biosystem 9050 Peptide synthesizer, using flow solid-phase peptide synthesis with Fmoc chemistry. This technique used preactivated Opfp amino acids with HOAt and preloaded PEG-PS resin. The polymer was purified by RP-HPLC on a Waters Delta Pack C18 A (300 × 39 mm ID). Purity was determined by RP-HPLC on a Dynamax C18 column (300 × 4.8 mm ID) equilibrated at a flow rate of 1 ml/min and eluted with a linear gradient from 5% to 80% CH₃CN (0.1% TFA) in 40 minutes The identity of the polymer was confirmed by TOF-MALDI MS (PerSeptive Biosystem). 

Preparation of Solutions

LTB₄ was dissolved in ethanol and diluted with HBSS (pH 7.3) to a final ethanol concentration of 0.001%. Synthetic peptides were dissolved in HBSS (pH 7.3). When necessary, the osmolality was adjusted between 280 and 320 mOsm by adding a small amount of distilled water. 

Neutrophil Isolation

These experiments followed the tenets of the Declaration of Helsinki and were approved by the Human Research Committee at Brookwood Medical Center. All donors signed written consent forms explaining the nature and possible consequences of the study. Blood was collected from only one donor each day. Following the technique of Ferrante and Thong, neutrophils were isolated from fresh heparinized human blood by centrifugation on Hypaque–Ficoll (density = 1.114), as previously described. ^{6 7} Isolated neutrophils (96%–99% viability) were resuspended in HBSS with 15 mM phosphate buffer at room temperature and gently agitated on a shaker. The purity of this cell suspension was ≥ 85% neutrophils and ≤ 5% mononuclear cells and platelets, with the remaining percentage consisting of red blood cells. Purified neutrophils were used in the polarization assay. All incubation mixtures were maintained between an osmolality of 280 to 320, a pH of 7.2 to 7.6, 15 mM phosphate buffer, and 50 μM Ca²⁺ and 50 μM Mg²⁺. 

Neutrophil Polarization Assay

The polarization assay was performed in a blind fashion. This assay was used to measure the neutrophil response to LTB₄ or synthetic peptides by determining the polarization index, a measure of the frequency and degree of cellular shape change after exposure to a chemoattractant. ⁸ Briefly, preincubated neutrophils (2 × 10⁵) were mixed with preincubated synthetic peptides in HBSS in a reaction chamber (total volume = 100 μl) at 37°C for 5 minutes. At the end of the incubation period, an aliquot was collected and mixed with an equal volume of 4% glutaraldehyde for microscopic observation. The remaining volume of each cell suspension was centrifuged immediately at 15,000g for 5 seconds to remove cells. The resulting supernatant was analyzed for lactic dehydrogenase (LDH) activity. ⁹ All incubations generated LDH activity, correlating with <5% cell death. Neutrophils in each sample were observed microscopically and assigned scores of 0 (resting = spherical cell with a smooth membrane), 1 (activated = irregular cell with uneven membranes), or 2 (polarized = cell length ≥ width × 2). Scores of 100 neutrophils for each sample were added and corrected by subtracting negative control values (neutrophils in HBSS only), which produced a polarization index. Bioactivity (EC₅₀) was expressed as the peptide concentration required to produce a neutrophil polarization index equal to 50% of the positive control (2 × 10⁻⁹ M LTB₄). Inhibition (ID₅₀) was expressed as the peptide concentration required to produce 50% inhibition of the EC₅₀ level of polarization. 

Results

Polarization Activity of Peptide Analogs of N-acetyl-PGP

Several peptide analogs of N-acetyl-PGP were synthesized and evaluated in the neutrophil polarization assay (Table 1) . The importance of the terminal amino acid residues was investigated first. Biological activity decreased in a systematic manner when the terminal amino acid(s) was omitted from the structure. N-acetyl-PG was sevenfold less active than N-acetyl-PGP, and activity was eliminated for the simple blocked proline derivative, N-acetyl-P. Simple dipeptides, GG, PG, or GP, and tripeptides, PGG and gly-pro-hyp, also were inactive. Two tripeptides, t-Boc-PGP and t-Boc-PGP-OMe, were synthesized to determine whether a free carboxyl terminus is needed for biological activity. Although t-Boc-PGP demonstrated sixfold less activity than N-acetyl-PGP, it did retain substantial activity. The tripeptide with blocking groups at both ends was inactive, suggesting that a free carboxyl group is needed for activity. These results demonstrated the importance of the terminal residues to induce neutrophil polarization. 

Next, we examined the importance of the N-acetyl group on biological activity. The unblocked tripeptide, PGP, was 30-fold less active than N-acetyl-PGP. Interestingly, the activity of the N-methyl derivative of PGP was comparable to the unblocked tripeptide. Apparently, this amino blocking group added little to the activity of the tripeptide. However, when a bulkier t-Boc group was used instead of the N-acetyl group to block the N terminus of the tripeptide, the activity of t-Boc-PGP was increased relative to PGP, but it was still sixfold less than the activity of N-acetyl-PGP. Taken together, the N-acetyl group provides an important structural component to N-acetyl-PGP; analogs with no or other N-terminal blocking groups have lower activity. 

Finally, we investigated if an extended PGP sequence could induce neutrophil polarization. A PGP polymer[ N-(PGP)₄-PGLG] containing 4 repeats of the PGP sequence was prepared. The activity of this peptide was 10-fold less than the simpler N-acetyl-blocked tripeptide, suggesting that these extended sequences are less effective than a shorter blocked PGP sequence. The most interesting discovery reported here deals with a pentapeptide, APGPR, containing the PGP sequence. This pentapeptide displayed activity equal to or slightly greater than N-acetyl-PGP. 

Inhibitory Properties of Synthetic Analogs

Synthetic analogs showing no detectable bioactivity were tested as potential inhibitors of N-acetyl-PGP–activated neutrophils. Gly-pro-hyp (ID₅₀ = 20 mM) was the only analog showing measurable inhibition when concentrations up to 70 mM were tested (Table 2) . 

Discussion

N-acetyl-PGP is generated directly by alkali degradation of corneal proteins. ³ The synthesis of peptide analogs of this chemoattractant identified several similar, new bioactive peptides for neutrophils and a number of inactive peptides. The small size of the N-acetyl-PGP molecule and the ability to manipulate the amino acid sequence lended itself to studies that differentiated those components of the chemoattractant responsible for its activity. This was accomplished by selecting specific groups on the N-acetyl-PGP molecule to add, substitute, and/or omit during synthesis of the peptide analog. 

For substantial bioactivity the analog required the presence of PGP alone or derivatives of PG in which the N-terminal proline is blocked. The presence of an acetyl- or t-Boc–blocking group on the N terminus of the tripeptide produces an uncharged amide at physiological pH. In contrast, PGP and N-methyl-PGP are very similar peptides in that both contain a positively charged amino N terminus at physiological pH. The favorable activity with a neutral amide elucidates a fuller understanding of the determinants of chemoattraction. This information is anticipated to prove useful in the structure-based design of antagonists. 

In this study APGPR, an activation pentapeptide for procolipase, produced equal or slightly greater polarization activity than N-acetyl-PGP. The potent activity of this agonist, containing the PGP sequence, gives promise that more agonists could be designed to further enhance biological activity. Pursuit of this line of investigation might be in the direction of other tetrapeptides or pentapeptides containing the PGP sequence but with an amide-protected N terminus, such as N-acetyl-APGPR. These findings might further studies of the inflammatory process and receptor recognition. 

The current approach generated one antagonist of neutrophil chemotaxis. High concentrations of gly-pro-hyp showed measurable inhibition of the chemoattractant. The potency of the gly-pro-hyp antagonist might be enhanced by further alteration of the molecule. A more feasible and powerful approach to secure a potent inhibitor might be the more time and cost efficient technology of using antisense peptides. ¹⁰ This technology has yielded promising results in preliminary studies. 

 Supported by National Eye Institute Grant EY04716. Mass spectrometry was performed by Marion Kirk at the UAB Comprehensive Cancer Center Mass Spectrometry Shared Facility, which is supported in part by a National Cancer Institute Core Research Support Grant P30CA13148. The mass spectrometer was purchased by funds from a National Institutes of Health Instrumentation Grant S10RR06487 and from UAB.

 Submitted for publication September 28, 1998; accepted November 6, 1998.

 Proprietary interest category: N.

 Corresponding author: Roswell R. Pfister, Suite 504, 513 Brookwood Boulevard, Birmingham, AL 35209. E-mail: [email protected]

Table 1.

 

 

Bioactivity of Synthetic Peptide Analogs

Table 1.

 

Bioactivity of Synthetic Peptide Analogs

Synthetic Peptides	EC50 (mM)
APGPR	0.34 ± 0.05
N-acetyl-PGP	0.5 ± 0.1
t-Boc-PGP	3.0 ± 2.3
[N-(PGP)4-PGLG]	3.0 ± 1.3
N-acetyl-PG	3.4 ± 2.2
N-methyl-PGP	15.0 ± 2.1
PGP	15.0 ± 3.6
N-acetyl-P	>40.0
GG	>40.0
PG	>40.0
GP	>40.0
PGG	>40.0
Gly-pro-hyp	>40.0
t-Boc-PGP-OMe	>40.0

 Values are means ± SD; n = 4 to 9. EC₅₀ was interpolated from dose–response curves of the neutrophil polarization index.

Table 2.

 

 

Inhibitory Properties of Synthetic Peptides

Table 2.

 

Inhibitory Properties of Synthetic Peptides

Synthetic Peptides	ID50 (mM)
Gly-pro-hyp	20.0 ± 5.6
N-acetyl-P	>70.0
t-Boc-PGP-OMe	>70.0
PG	>70.0
PGG	>70.0
GP	>70.0
GG	>70.0

 Values are means ± SD; n = 4. Neutrophils were activated with 500 μM N-acetyl-PGP. ID₅₀ was interpolated from dose–response curves of the polarization index.

 

Amino acid analysis was performed by Kelly Morrison at the UAB Glycoprotein Analysis Core Facility.