structure determination of a human lymphocyte derived neutrophil activating peptide (lynap)

Vo1.151, No. 2,1988

March 15,1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 883-890

STRUCTURE DETERMINATION OF A HUMAN LYMPHOCYTE

DERIVED NEUTROPHIL ACTIVATING PEPTIDE (LYNAP)

Harry Gregory, Janice Young, Jens-M. Schr~der*,

Ulrich Mrowietz* and Enno Christophers*

ICI, Pharmaceuticals Division, Mereside Alderly Park

Macclesfield Cheshire SK 10 4TG, Great Britain

*Dept. of Dermatology, University of 2300 Kiel,

Schittenhelmstr. 7, FRG

Received January 27, 1988

Summary Phytohemagglutinin or Concanavalin A-stimulated human T-lymphocytes produce a factor (LYNAP) with potent chemotactic and enzyme degranulating activity in peripheral human neutrophils. Sequence analysis of LYNAP established an apparently novel 72 residue polypeptide structure. Examination of protein data bases showed that LYNAP had about 30 % sequence homology with recently characterised connective tissue activating proteins produced by platelets. Furthermore, it was subsequently found that the amino acid sequence is large- ly the same as that predicted from a cDNA clone derived from mRNA elevated in peripheral human leukocytes stimulated by mi togens. ® 1988 Academlc Press, Inc.

In recent years several reports indicate that human lympho-

cytes are able to produce factors activating neutrophil che-

motaxis when stimulated with mitogens like phytohemagglutinin

(PHA) or Concanavalin A (Con A) (I-3). So far, a detailed

biochemical characterization of these lymphokines has not

been performed.

Abbreviations: HPLC, high performance liquid chromatography; LYNAP, lymphocyte derived neutrophil activating peptide; MONAP, monocyte derived neutrophil activating peptide; PMNL, polymorphonuclear leukocyte; PTH, phenylthiohydantoin; RP, reversed phase; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid.

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0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc.

All rights of reproduction in any form reserved.

Vol. 151, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In a recent study, we were able to isolate and purify a po-

tent neutrophil chemotactic polypeptide from PHA- or Con A-

stimulated human T-lymphocyte preparations (4). This factor

has been found to be homogeneous by several criteria, giving

a single and sharp peak upon reverse phase HPLC as well as

a single line at 10 kD upon SDS-PAGE under nonreducing con-

ditions (4).

In the present study we describe amino acid sequence analy-

sis of this neutrophil chemotactic factor.

MATERIALS AND METHODS

Lymphoc_~p_y_~e purification: Human mononuclear cells were separated from venous blood by Ficoll centrifugation as recently described (4). Monocytes were separated by adherence to fibro- nectin coated plastic dishes according to Freundlich and Av- dalovic (5). Nonadherent cells were further purified by a modification of the procedure of Eisen et al (6) using nylon wool filtration followed by 15 min. incubation in plastic petri dishes. The final lymphocyte preparation contained 88 ~ 2 % T lymphocytes (Leu I Pan T monoclonal antibody, Becton-Dickinson, Heidelberg, FRG), 1% B-lymphocytes (Dako Pan B monoclonal antibody) and <I % monocytes (Serotec, FMC 32 monoclonal antibody). Bioassays: Chemotaxis as well as release of lysosomal enzymes ~marker-enzyme: myeloperoxidase) by human neutrophils was estimated as recently described using the Boyden chamber tech- nique and the cytochalasin B pretreatment-assay, respectively (7). Production of the lymphocyte d_e_erived neutrophil activatin~ peptide: Purified human lymphocyte preparations were suspen- ded in RPMI-medium and incubated in plastic tissue culture flasks (Falcon) at 37 ° C after adding PHA (101ug/ml) or Con A (10/ug/ml) for 20 - 40 h.

Purification of the lymphocyte PMNL-activating factor (LYNAP): Purification was performed as described in detail elsewhere (4). Briefly, culture supernatants were concentrated on an Amicon YM-5 membrane and separated by gel permeation chromatography using a 2.6 x 75 cm G-75 gel column (Pharmacia, Uppsala) with 0.05 M ammonium formate, pH 4 as eluent. Biologically active fractions were further purified by HPLC techniques using wide pore reverse phase (RP-8) HPLC (Zorbax PEP-RP-I, Dupont, Bad Nauheim, FRG) with a gradient of acetonitrile containing 0.1% (v/v) TFA followed by size exclusion HPLC with a 0.8 x 60 cm TSK-2000 SW column (LKB, Bromma, Sweden) and 0.1% TFA as eluent. As final purification step narrow pore reverse phase-HPLC (Nucleosil RP-18, 5zu , Merck, Darmstadt, FRG) was used with an acetonitrile gradient for elution of the chemotactic peptide. Polyacrylamide gel electrophoresis: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for peptides was performed as described (4,7) in

884


the presence of 8M urea using the method of Swank and Mun- kres (8). Proteins were visualized by silver staining. Protein determinations: During HPLC separations absorbance was monitored at 215 nm. Integration values obtained by the peak integrator were converted to protein concentrations using~cytochrome C for cali- bration as described (7). 0.7 - I x 10 ~ integration units corresponded to I/ug protein. Preparations of carboxamidometh~l-derivative~ The purified LYNAP (60/ug in 30~ul water) was mixed with 6 M guanidine hydrochloride, 0.1M ~ris hydrochloride pH 8.5 (300/ul). The tube was thoroughly flushed with nitrogen and then dithiothreitol was added (5/ul of a 50 mg/ml solution in the guanidine buffer). After flushing again the tube was sealed and kept for 4 hrs at 37 ° C. Iodoacetamide was added (5 mg in 50/ul buffer) and the nitrogen flushed tube kept at ambient temperature in the dark for I hr. The mixture was applied directly to a C18 HPLC column (Vydac) and eluted with a linear gradient of 0.1% trifluoroacetic acid in water (A) to 0.1% trifluoroacetic acid (B) in acetonitrile. The single derivative peak eluted with 40 % of the acetonitrile solution. The appropriate fractions were combined and dried by centri- fical evaporation. Amino acid analysis: The carboxamidomethyl derivative (20/ug) was hydrolysed in vacuo with 6Nohydrochloric acid containing 1% phenol for 16 hrs at 110 C. The dried hydrolysate was analysed under standard conditions using an LKB.4400 amino acid analyser. Amino acid sequence_analysis: The intact protein and subsequent enzymically derived fragments were analysed using an Applied Biosystems gas phase sequencer model no. 470A with on line HPLC analysis of the PTH derivatives, model no. 120A. Chymotryptic digestion: Carboxamidomethyl-LYNAP (7/ug) was taken into 0.1M ammonium- bicarbonate, 0.004 M calcium chloride (50/ul) and treated with chymotrypsin-Worthington (10/ul from a ~51ug/ml solution in bicarbonate). The solution was kept at 37 C for 2.2 hrs and after acidification was applied directly to a reverse phase HPLC column (Vydac C18). A gradient of 5 % B to 60 % B as above was applied over 60 min at a flow rate of Iml/min and fractions of 0.5 ml were collected. Appropriate fractions were combined, dried and applied directly to the sequencer. Six peptides were fully sequenced. Tryptic digestion: Carboxamidomethyl-LYNAP (20/ug) was ~aken into the bicarbonate buffer (70/ul) and warmed to 37 C. Trypsin-Worthing- ton (10/ul of a 10/ug/ml solution) was added and after 30 mins at 37 ° C the contents of the tube were diluted with 0.1 % aqueous trifluoroacetic acid and applied to the HPLC system as above. Eight of the peptides produced were sequenced.

RESULTS AND DISCUSSION

The sequence shown in Fig. I is derived from the direct se-

quencing which was interpretable as far as residue 37. How-

ever, sequencing of the parent molecule indicated two spe-

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CTAP-III NLAKGKEE SLD.~D ~ v ~ { F r ~ . r , . . ~ : ~ T ~.L,?,,~ ,~.K.~.VT~p~TV~,T,rO~VL~*O c ' . , - . . . . . . ,

, ~ , ,~..~ .'., , PF-4 E'.. :,.u,:6..c:'~.~"-~::..P#.i..# .:.d',~e, ::.. f: ~.~D~'c~'::: .... '~ ~,""

MONAP ~. :'..' "{.:' C':.0 ;~' '~'%9':" ~...pv "14 'L~"¢ :. ':G.: ~'C' "~ ' .,~

• q ", ' ~i, : "'•' "

LYNAP 3.-.

T1 T2 C2 T6 C3 I8 C1 T4 "113 T7 C6

T3 C4 C5

Figure I Complete amino acid sequence of a lymphocyte derived neutrophil activating peptide (LYNAP). For comparison, the amino acid sequences of a connective tissue activating peptide (CTAP-III) (9), platelet factor 4 (PF-4) (10), a monocyte derived neutrophil activating peptide (MONAP) (N-terminal sequence only) as well as the deduced amino acid sequence of a ~-thromboglobulin-like protein (3-10 C) predicted from cDNA clones from human blood leukocytes (11) are shown. In addition, alignments of peptides isolated by chymotryptic (CI-C6) as well as tryptic digestion peptides (TI-T8) of car- boxyamidomethylated LYNAP are indicated. The arrow shows N- terminal sequence of intact LYNAP up to residue 37 as well as N-terminal sequences of some cleaved peptides. In brackets the sequence of the additional N-terminal pentapeptide is demonstrated. In the predicted sequence of the hypothe- sized peptide corresponding to the ~-thromboglobulin-like protein 3-10 C the sequence of the signal peptide is under- lined. Dark areas indicate homology between all five peptides shown.

cies to be present, one of which (approx. 30 % of the pre-

paration) contained an additional ~-terminal pentapeptide.

Thereafter overlap of the chymotryptic and tryptic fragments

(Fig. 2 and Fig. 3) provided unequivocal evidence of the

structure as shown in Fig. I.

The purified LYNAP converted to a single carboxamidomethyl-

derivative with similar amino acid composition to the star-

ting material (not shown) indicating a single polypeptide

chain was involved. Direct sequence analysis of the underi-

vatised material gave a run of 37 amino acid residues. Enzy-

mic digests produced several apparently pure peptides (Fig.

2 and Fig. 3) appropriate for further sequencing. Those re-

levant to establishing the full structure are shown in Fig.

I. Peptide CI, for example, showed the two Cys residues as

recognisable carboxamidomethyl PTH derivatives whereas in

886

VoI. 151, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0

C4 C2 C3 C5 C1 C6

v i =

40 30 20 10

RETENTION TIME [min.]

fl-4

Figure 2 Chymotryptic digest of carboxamidomethyl-LYNAP separated by HPLC on a reverse phase (C 18) column. Peptides used for amino acid sequence analysis are indicated (CI C6).

.=_

gll

the intact molecule Cys is inferred only by the absence of

a PTH residue. The chymotryptic peptides from cleavage after

aromatic residues (or leucine) and the tryptic peptides from

cleavage after basic residues provided unambiguous evidence

C~ O

T7 T5T4 T3 T1 T2 T6

- 60

, , - / H

40 30 20 10 0

R E T E N T I O N TIME [min,]

Figure 3 Tryptic digest of carboxamido-methyl-LYNAP separated by HPLC using the same reverse phase column as indicated in Fig. 2. Peptides used for amino acid sequence analysis are indicated (TI T8).

-60

'0

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for the remaining part of the structure. Although no overlap

was found between residues 47-48 (K-L) in the peptides selected,

the certainty of the other overlaps made 47-48 also quite

clear cut.

The amino acid composition from the sequence was in keeping

with that observed from the single amino acid analysis. The

ratios were calculated on the basis of a 72 residue peptide,

so that partial contribution of the N-terminal pentapeptide

gave higher ratios for the residues indicated. Valine and

isoleucine figures were lower but this is entirely predict-

able from a relatively short hydrolysis, particularly when

val-val and ileu-ileu sequences are involved. Tryptophan was

not seen in the hydrolysate and its position derived entire-

ly from the peptide sequencing.

When the structure was established, examination of the NBRF

protein database (version 11.0) revealed sequence similarity

to two proteins related to connective tissue activating pro-

teins both secreted by platelets (9,10). The correspondence

(Fig. I) amounts to 24 residues commonly located but LYNAP

does have an extra amino acid inserted at position 17, where-

as both the others have a single lipophilic residue at this

point. The distribution of conserved residues across the

three molecules implies similar spatial structures which may

be reflected in their biological properties.

Recently, Schmid and Weissmann (10) described two cDNA clones

corresponding to mRNA that is present in mitogen induced

human leukocytes. The deduced amino acid sequence of one of

these clones, 3-10 C, shows homologies to connective tissue

activating peptide-III (CTAB-III) as well as platelet factor

4 (PF-4, Fig. I). A surprising result, however, is the com-

888


Table I ,

Amino acid composition of human carboxamidomethyl-LYNAP and MONAP. The amino acid ratios (residues/molecule) from a single analysis of a hydrolysate are given. The subsequent integral values from sequencing are presented in brackets with those noted for the N-terminal pentapeptide given as additions.

Amino acid LYNAP MONAP Amino LYNAP MONAP acid

CM Cys 3.6 (4) n.d. Val 4.1 (5+I) 4.0 Asx 5.3 (5) 6.1 Ile 2.7 (5) 3.2 Thr 2.3 (2) 2.4 Leu 6.7 (6+I) 6.4 Ser 5.2 (5) 4.7 Tyr 0.9 (I) 1.0 Glx 10.1 (10) 10.0 Phe 2.7 (3) 2.8 Pro 5.1 (4+I) 6.5 His 2.1 (2) 1.9 Gly 3.2 (2) 2.6 Lys 8.8 (9) 8.4 Ala 4.7 (3+I) 3.6 Arg 4.9 (4+I) 4.3

** Neither methionine nor tryptophan residues were detected. Not detected in the native material.

plete homology of LYNAP with the predicted amino acid sequence

of 3-10 C starting at amino acid 28 (serine) (Fig. I). More-

over the LYNAP sequence extended by 5 amino acid residues

at the N-terminus, found as a minor component with LYNAP,

showed complete homology to the predicted peptide correspon-

ding to 3-10 C-cDNA (10), except for the predicted signal

peptide (22 amino acids). This indicates that LYNAP is a

cleavage product of this precurser molecule cleaved between

residue 5 and 6 (arginine and serine).

In addition, a monocyte derived neutrophil activating pep-

tide (MONAP) which we recently isolated from LPS-activated

human monocytes and purified to homogeneity (7) demonstrates

exactly the same N-terminal sequence as the lymphocyte derived

chemotaxin (Fig. I). The amino acid composition at underi-

vatised material given in Table I provides confirmation of

the structural identity of LYNAP and MONAP - two potent neu-

trophil chemotactic agents produced by entirely different

blood cells.

889


ACKNOWLEDGEMENTS

This work was supported in part by DFG grant Schr 305/I-I. We are gratefully indebted to Miss C. Gerbrecht, Miss A. StHtzl-Studt and Miss. E. Ohm for their excellent technical assistance and Mrs. I. B6ttjer for typing the manuscript.

REFERENCES

I. Rocklin, R.E., MacDermott, R.P., Chess, L., Schlossman, S.F., and David, J.R. (1974) J. Exp. Med. 140, 1303 - 1311.

2. Ward, P.A., Remold, H.G., and David, J.R. (1969) Cell. Immunol. I, 162 - 174.

3. Altman, L.C., Chassey, B., and Mackler, B.F. (1975) J. Immunol. 115, 18 - 21.

4. SchrSder, J.-M., Mrowietz, U., and Christophers, E. (1987), submitted for publication.

5. Freundlich, B., Avdalovic, N. (1983) J. Immunol. Methods 52, 31 - 37.

6. Eisen, S.A., Wedner, H.J., and Parer, C.W. (1972) Immunol. Comm. I, 571 - 579.

7. SchrSder, J.-M., Mrowietz, U., Morita, E., and Christophers, E. (1987) J. Immunol. 139, 3474 - 3482.

8. Swank, R.T., and Munkres, K.D. (1971) Anal. Biochem. 3, 462 - 470.

9. Castor, C.W., Miller, J.W., and Walz, D.A. (1983) Proc. Natl. Acad. Sci. USA, 80, 765 - 769.

10. Denel, T.F., Keim, P.S., Farmber, M., and Heinrikson, R.L. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2256- 2248.

11. Schmid, J., and Weissmann, C. (1987) J. Immunol. 139, 250 - 256.

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structure determination of a human lymphocyte derived neutrophil activating peptide (lynap)

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