Purification and characterization of neutrophil chemotactic factors of Streptococcus sanguis

Biochimica et Biophysica Acta, 758 (1983) 181-186 Elsevier

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BBA 21500

PURIFICATION AND CHARACTERIZATION OF NEUTROPHIL CHEMOTACTIC FACTORS OF STREPTOCOCCUS SANGUIS YOICHIRO MIYAKE a, TADASHI YASUHARA b, KAZUHIRO FUKUI a,., HIDEKAZU SUGINAKA NAKAJIMA b and TAKAFUMI MORIYAMA b,**

a, TERUMI

a Department of Microbiology, Hiroshima University School of Dentistry, 1 -2- 3 Kasumi, Minami-ku, Hiroshima 734 and h Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, 2- 3-10 Kanda Surugadai, Chiyoda- ku, Tokyo 101 (Japan) (Received December 24th, 1982) (Revised manuscript received April 28th, 1983)

Key words: Neutrophil; Chemotaxis," (S. sanguis)

Two neutrophil chemotactic factors were isolated from the culture filtrates of Streptococcus sanguls ATCC 10556 and were chemically characterized as N-terminal blocked peptides of low molecular weight. One of the factors consisted of proline, valine, methionine, isoleucine and leucine and the other of methionine, isoleucine, leucine and phenylalanine. In both factors, methionine was detected as the sole N-terminal amino acid, but the amino group was blocked. The removal of N-terminal methionine yielded several N-terminal amino acids, suggesting that S. sanguis produced several N-terminal blocked methionyl peptides, all of which could be chemotactically active.

Introduction

The formation of dental plaque is essential for the initiation and development of inflammation in the periodontal tissue [1-3]. A mechanism by which dental plaque can induce inflammation is the attraction of neutrophils and macrophages into the gingival crevice and gingival tissue. In fact, neutrophils are the principal cells of the gingival crevicular and pocket exudates [4-6]. Continuous emigration of such inflammatory cells could lead to tissue destruction [7,8]. It is known that dental plaque shows chemotactic ability on neutrophil [9,10]. Plaque bacteria

* Present address: Department of Microbiology, Okayama University School of Dentistry, 2-5-1, Shikata-cho, Okayama 700, Japan. ** Present address: Department of Microbiology, AichiGakuin University School of Dentistry, 2-1 l, Suemori-dori, Chikusa-ku, Nagoya 464, Japan. 0304-4165/83/$03.00 © 1983 Elsevier Science Publishers B.V.

is one of the components which are responsible for chemotactic ability [ 11,12]. Most bacteria elaborate chemotactic factors, which, however, have not been well characterized. Ward et al. [13] demonstrated by gel filtration technique that chemotactic factors produced by various strains of staphylococci, streptococci, pneumococci and others were substances of small molecular weight, but did not chemically define them. Schiffmann et al. [14] reported the isolation and characterization of neutrophil chemotactic factors from Escherichia coti and described them to be small molecular weight peptides containing aspartic acid, serine, glutamic acid, alanine and glycine. With the above in mind, it was considered important to characterize chemotactic factors released from plaque bacteria. We have tested a total of 19 strains of 12 species of oral bacteria with respect to chemotactic ability [ 15]. Very few of the organisms tested did not elaborate any chem-

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otactic factor. Since S. sanguis was one of the most active strains among them and the bacterial species commonly found in the gingival crevicular area, we decided to isolate and chemically characterize its neutrophil chemotactic factor. Materials and Methods

Culture of S. sanguis. S. sanguis ATCC 10556 was grown in Carlsson's synthetic medium for S. sanguis [16]. Table I shows the chemical composition of this medium. 500 ml preculture was inoculated into 4.5 1 medium and incubated at 37°C with stirring to a late log phase. The suspension was then centrifuged at 5500 × g for 15 rain at 4°C. 170 1 supernatant was filtered through a Millipore filter (0.45/~m, Millipore Corp., U.S.A.) and used as starting material. Assay of chemotactic activity. The agarose plate method was employed [17,18]. 10 ml heparinized peripheral blood from healthy human volunteers was added to 3 ml 6% dextran in normal saline to precipitate red blood cells. The resulting supernatant was layered over 3 ml Lymphoprep (Nyegaad, Norway) and centrifuged at 400 × g for 30 rain at 20°C. The precipitates were suspended in distilled water to lyse the residual red blood cells and an equal volume of double-strength saline was added to restore isotonicity. Neutrophils thus obtained were washed with medium 199 (Grand Island Biological Company, U.S.A.) and resuspended in the same medium to give a concentration of I • 108 cells/ml. 5 ml medium 199 containing 1% agarose, 10% heat-inactivated fetal calf serum (Grand Island Biological Company, U.S.A.) and 375 # g / m l sodium bicarbonate was hardened in a plastic petri dish. Three serial wells, whose diameters and distances from each other were 2.3 mm each, were cut with a stainless steel cutter. 5 ttl aliquots of neutrophil suspension were poured into the center well, 5 #1 each of samples to the outer wells and 5 /d each of the control solution (usually medium 199) to the inner wells. The plate was incubated at 37°C in humidified air containing 5% CO 2 for 2 h. After incubation, cell migration distances from the center well toward sample (A) and toward control (B) were measured with a stereoscopic microscope (Type MTD,

TABLE I C O M P O S I T I O N OF C A R L S S O N ' S S Y N T H E T I C M E D I U M F O R S. S A N G U I S Components Alanine Arginine-HC1 Asparagine Aspartic acid Cysteine-HC1 Cystine Glutamic acid Glycine Histidine-HCl Hydroxyproline Isoleucine Leucine Lysine-HC1 Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Adenine Guanine Uracil Folic acid Biotin p-Aminobenzoic acid Thiamine-HC1 Riboflavin Pyridoxine-HCl Ca-pantothenate Nicotinic acid MgSO 4 • 7H 2° FeSO 4 • 7H 2° MnSO4.4H20 N aCl CH 3COONa. 3 H 2 0 CHaCOONH 4 Glucose Potassium phosphate buffer (pH 7.0)

Concentration ( m g / l ) 100 100 200 100 50 50 500 200 200 100 100 100 100 200 100 100 100 100 100 100 100 10 10 10 0.005 0.0025 0.1 0.5 0.5 1.0 0.5 1.0 200 l0 l0 l0 10000 100 l 0000 0.1 M

Nikon, Japan). Chemotactic activity was expressed as chemotactic differential (A-B). In the case that the front line of migrating leukocytes appeared not peaked but flat due to higher activity of the sample, we regarded the chemotactic differential of this sample to be over 2.0 mm. Purification of active fraction. A flow chart of

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purification procedures is presented in Fig. 1. Every 30 1 of culture filtrate was applied to an Amberlite XAD-2 column (700 x 1600 mm, R o h m and Haas Corp., U.S.A.), washed with distilled water followed by 5% methanol, and then eluted with 99% methanol. Methanol was evaporated and the dried residues were dissolved in 100 ml aqueous phase of c h l o r o f o r m / m e t h a n o l / w a t e r (45 : 60 : 40, v / v ) , and every 10 ml was subjected to droplet countercurrent c h r o m a t o g r a p h y with c h l o r o f o r m / m e t h a n o l / w a t e r (45 : 60 : 40, v / v ) . The column consisted of 24 silicon coated glass tubes (4 x 550 m m each) which were supported vertically. The top end of each tube was connected with a Teflon tube to the bottom end of the next tube. We adopted the ascending method. The column was filled with the chloroform phase as the stationary phase and the sample which was dissolved in the aqueous phase was injected from the bottom end of the first tube. The column was eluted with 60 ml of the aqueous phase with appropriate pressure to form droplets of the aqueous phase. Finally the residual stationary phase was pushed out with air. 5 ml fractions from the top end of the last tube were collected. The active fraction in the chloroform phase was extracted with aqueous ammonia

Culture filtrate 170 liter

I

XAD-2 column chromatography Active fractions were dried and dissolved in 100 ml aqueous phase Dro let countercurrent chromatography Active principle was extracted with aqueous ammonia from chloroform phase Dried and dissolved in 6.0 ml 90% methanol containingb.2% acetic acid Seph adex LH-20 column chromatography Main active fractions were dried and dissolved in 3.0 ml 0.01 M HCI SP-Sephadex C-25 column chromatography Main active fractions were dried and dissolved in 2.0 ml distilled water Sephadex G-10 column chromatography l Fraction I (fractions 13-14) 4.0 ml Fraction II (fractions 15-17) 6.0 ml Sep adex G-10 column rechromatography Fraction I (fractions 14-15) 4.0 ml Fraction II (fractions 17-18) 4.0 ml Fig. 1. Flow chart of purification procedures.

at p H 10.0. The extract was dried and dissolved in 6.0 ml 90% methanol containing 0.2% acetic acid. Every 1.0 ml methanol solution was applied to a Sephadex LH-20 column (8 x 900 mm, Pharmacia Fine Chemicals, Sweden) equilibrated with 90% methanol containing 0.2% acetic acid and eluted with the same solvent. 2 ml fractions were collected and dried, respectively. A part of each fraction was dissolved in distilled water and assayed for chemotactic activity. The active fractions from Sephadex LH-20 were combined, dissolved in 3.0 ml 0.01 M HC1 (pH 2.0), and then chromatographed on SP-Sephadex C-25 with 0.01 M HC1 (pH 2.0) as eluting solvent. 2 ml fractions were collected. The active SP-Sephadex fraction, which was not adsorbed, was dried and dissolved in 2.0 ml distilled water. Every 1.0 ml was applied to a Sephadex G-10 column (9 x 920 mm) equilibrated with distilled water and 2.0 ml fractions were collected. Two active fractions eluted with distilled water were rechromatographed on Sephadex G-10 (9 x 920 mm). The rechromatographed fractions were diluted 100times with distilled water and assayed for chemotactic activity. The active fractions were lyophilized and stored at - 2 0 ° C . Enzymatic treatment of active fractions. The enzyme solutions used were as follows: carboxypeptidase A (Worthington Biochemical Corp., U.S.A.) dissolved in 10% LiC1 at 1.0 m g / m l and carboxypeptidase P (Protein Research Foundation, Japan) dissolved in acetic acid (pH 5.0) at 1.0 mg/ml. 5/~1 carboxypeptidase A was added to samples dissolved in 50 /~1 0.1 M triethylamine (pH 8.3). When samples were digested with carboxypeptidase P, samples were dissolved in 50/tl acetic acid (pH 5.0). The mixtures were incubated at 37°C for 12h. Dansylation of peptides. 0.5-5 nmol peptides were dissolved in 5 /tl 0.1 M triethylamine (pH 8.3) to which 5/~1 0.5% dansyl chloride in dioxane was added. The mixtures were incubated at room temperature for more than 3 h. After incubation, the mixtures were heat-dried under a nitrogen stream.

Methionine degradation with cyanogen bromide. A volume of 50-100/~1 10 m M cyanogen bromide in 70% formic acid was added to a dried sample

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and the tube was sealed. After incubation at room temperature overnight, 1 ml distilled water was added to the tube and the sample was then lyophilized. Amino acid analysis. 1-5 nmol peptides were hydrolysed with 6 M HC1 at 110°C for 24 h. The amino acid composition was determined by an amino acid analyzer JOEL 5-AH (ninhydrin method) or TOYO-SODA HLC-805 (orthophthalaldehyde method). Thin-layer chromatography. H P T L C Fertigplatten Kieselgel 60 (Art. 5631, Merck, F.R.G.) was used for thin-layer chromatography (TLC) of peptides with c h l o r o f o r m / m e t h a n o l / a c e t i c acid (15 : 4 : 1, v / v ) or butanol/acetic a c i d / w a t e r (4 : 1 : 5, v/v). Polyamide layer (Cheng Chin Trading Company, Taiwan) was used to identify dansylated peptides with benzene/acetic acid (9 : 1, v / v ) or 1.5% formic acid. Fluorescamine reaction. 0.01% fluorescamine (F. Hoffmann-La Roche & Co. AG., Switzerland) in acetone was sprayed on TLC plate. Fluorescamine positive spots were visualized by ultraviolet light at 360 nm.

~__aqueous -~ E

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1.o

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d

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i

i

2o 30 fraction number

Fig. 2. Elution profile of Amberlite XAD-2 methanol eluate in droplet countercurrent chromatography. The column consisted of 24 glass tubes (4 x 550 m m each), which were connected to each other with Teflon tubes, and supported vertically. The solvent system was c h l o r o f o r m / m e t h a n o l / w a t e r (45:60:40, v/v). An ascending method was employed. Fractions of 5 ml each were collected and assayed for chemotactic activity.

were rechromatographed on Sephadex G-10 (Fig. 6). We subsequently employed both DEAE-Sephadex column chromatography and reverse phase high-pressure liquid chromatography. However, in both methods, the active fraction appeared as a single peak and contained at least four bands which were detected on TLC.

Properties of Sephadex G-IO fractions I and II.

Results

Purification. The culture filtrate of S. sanguis demonstrated chemotactic activity of 0.7 mm as chemotactic differential, while Carlsson's synthetic medium itself had no activity. Chemotactic factors in the culture filtrates were well adsorbed to Amberlite XAD-2, and eluted with methanol. In this procedure, samples were desalted and, at the same time, concentrated. In droplet counter-current chromatography, the major active fraction was found in the stationary (chloroform) phase (Fig. 2). The aqueous ammonia extract of the major fraction was applied to Sephadex LH-20 column chromatography. Two active fractions were isolated, one with a very high activity (Fig. 3). When the major active fraction from Sephadex LH-20 was subjected to SP-Sephadex C-25 chromatography one major and two minor active fractions were obtained (Fig. 4). A little activity was eluted by changing the eluent, HC1, to aqueous ammonia. The major active fraction from SP-Sephadex was further separated into two fractions (fractions I and II) by Sephadex G-10 (Fig. 5). These fractions

d L chl°r°f°rm

phase ~I

Both fractions I and II from Sephadex G-10 column were stable to heating at 100°C for 15 rain. These fractions were analyzed for amino acid composition. Fraction I contained proline, valine, methionine, isoleucine and leucine, while fraction II contained methionine, isoleucine, leucine and phenylalanine. By treatment with carboxypeptidase A, fraction I lost 70% of its activity and fraction II was completely inactivated. F-Met-Leu20 E "5

~1 : 3

1.0

8

go

10

20 30 fraction number

Fig. 3. Gel filtration of the sample from droplet countercurrent chromatography on Sephadex LH-20. The column ( 8 × 9 0 0 m m ) was eluted with 90% methanol containing 0.2% acetic acid.

185 2.0 E

1.0 0

go

10

20 30 &O fraction number

Fig. 4. SP-Sephadex column chromatography of the major active fraction from Sephadex LH-20. The sample was applied to SP-Sephadex C-25 column (10x260 ram) in 0.01 M HCl (pH 2.0). The arrow indicates the start of elution with aqueous ammonia (pH 10.0). 2 ml fractions were collected.

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Fig. 5. Gel filtration of the major active fraction from SP-Sephadex on Sephadex G-10. The major active fraction from SPSephadex was chromatographed on Sephadex G-10 column (9 x 920 mm) with distilled water. 2 ml fractions were collected. The bars indicate the distribution of fractions I and II.

2.0

Fraction I

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~

Fraction IT

H

1.0

0

' 10

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Fig. 6. Rechromatography of Sephadex G - I 0 fractions I and I I .

For experimental conditions see legend to Fig. 5. The fractions indicated by bars were used in the analysis.

Phe (Protein Research Foundation, Japan) used as control was completely inactivated by carboxypeptidase A. Fraction I was, however, completely inactivated by carboxypeptidase P, which was also capable of breaking the peptide bond adjacent to the praline residue. Amino acid analysis after digestion by carboxypeptidase A revealed all the foregoing amino acids except for methionine. Fractions I and II were negative for fluorescamine reaction. However, after degradation of methionine by cyanogen bromide, both fractions became positive. The cyanogen bromide treated samples of both fractions I and II were separated into at least four bands on TLC, and at least four N-terminal amino acids were detected by the dansylation method. Fractions I and II lost 64 and 87% of their activities after treatment with cyanogen bromide, respectively. Both fractions became fluorescamine reaction positive after treatment with 1 M HC1. Fractions I and II lost 76 and 78% of their activities by treatment with 1 M HCI, respectively. Although the treated fractions were separated into at least four bands on TLC, methionine was detected as a sole N-terminal amino acid. Discussion

Two kinds of neutrophil chemotactic factors were purified from the culture filtrate of S. sanguis ATCC 10556. The final Sephadex G-10 fractions (fractions I and II), which were not excluded from this gel, were of lower molecular weigh and heatstable. For further purification, we used either DEAE-Sephadex column chromatography or reverse phase high-pressure liquid chromatography, but these were not successful. The active principles involved in these fractions seemed to be peptides, because they lost their chemotactic activities by carboxypeptidases. The principles were not adsorbed to SP-Sephadex, a cation exchanger, and were negative for the fluorescamine reaction. These facts suggested that the N-terminal groups were blocked. The fractions were adsorbed to DEAESephadex, an anion exchanger (data not shown), suggesting that the terminal carboxy groups were free. After digestion of fractions I and II with carboxypeptidase A, methionine was not detected, although they contained methionine. After treatment with 1 M HC1, they became fluorescamine

186

reaction positive, and the N-terminal amino acids of the treated samples were methionines. These results suggested that the N-terminal amino acids of fractions I and II were methionines, and the amino groups of the methionines were blocked. However, the blockage was so weak that treatment with 1 M HC1 could make the amino group free. This blockage is important, because the treatment with 1 M HC1 destroyed the chemotactic activities of fractions I and II. After removal of methionine with cyanogen bromide, four or more N-terminal amino acids were detected, suggesting that fractions I and II contained several peptides, all of which had methionine as N-terminal amino acid. Schiffmann et al. [14] characterized the chemotactic components from E. coli and found that they were small, heterogeneous peptides with blocked terminal amino groups. However, since the low levels of attractants hampered further purification, Schiffmann et al. [19] synthesized Nformylmethionyl peptides. Most of them showed rather high activities. The idea was due to the fact that formylmethionyl peptides split from bacterial nascent proteins [20,21] seemed to be involved in bacterial culture fluids as a class of compounds characteristic of the bacterial cells. It is interesting that the chemotactic peptides isolated and characterized in the present study were different from those of E. coli culture filtrates [14] in terms of amino acid composition and resemblant to some artificially synthesized N-formylmethionyl peptides, such as f-Met-Leu and f-Met-Leu-Phe [22]. However, the final preparations of chemotactic factor remained still heterogeneous, as judged from TLC patterns. Each of them consisted of several N-terminal blocked methionyl peptides. However, in view of the above consideration, most, if not all, of these peptides seemed to be chemotactic. We started to purify the factors from 170 1 of the culture filtrate, and due to the low levels and high specific activities of the factors, it was difficult to proceed further. However, as far as amino acid composition is concerned, our results support the idea of Schiffmann et al. [19] that bacterial chemotactic factors might be split product from nascent proteins [20,21]. Although chemotactic factors from some other bacteria must be also examined, it seems valid to employ synthetic peptides as a tool to evaluate the

role of bacterial chemotactic factors in periodontal tissue damage. Binding of labeled f-Met-Leu-Phe to receptors on neutrophils of juvenile periodontitis patients has been examined [23].

Acknowledgement This work was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Japan. (Project No. B-148289, C357551, 57771192.)

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