THE JOURNAL 0 1991 by The American Society for Biochemistry and Molecular Biology,

OF BIOLOGICAL CHEMISTRY Inc.

VOl. 266, No. 8, Issue of March 15, pp. 4713-4718,1991 Printed in U.S.A.

Demonstration of Peptidoglycan-binding Sites on Lymphocytes and Macrophages by Photoaffinity Cross-linking*

(Received for publication, August 22, 1990, and in revised form, October 24, 1990)

Roman Dziarski From the Northwest Center for Medical Education, Indiana Uniuersity School of Medicine, Gary, Indiana 46408

One dominant binding site (70 kDa 6.5 PI protein) for bacterial cell wall peptidoglycan (PGN), a macro- phage activator and polyclonal B cell mitogen, was demonstrated on mouse B and T lymphocytes and mac- rophages by photoaffinity cross-linking and two-di- mensional polyacrylamide gel electrophoresis. This binding site was not present on erythrocytes. The bind- ing was specific for polymeric PGN and was competi- tively inhibited by unlabeled PGN with ICs0 = 48 pg/ ml (0.38 p ~ ) . The binding was partially inhibited by 0-acetylated PGN monomers (IC60 = 469 pg/ml, 521 NM), dextran sulfate (ICso = 1024 pg/ml, 124 p ~ ) , and (GlcNAc)3 (ICso = 6.6 mg/ml, 10 mM), and was not inhibited by non-0-acetylated PGN monomers and di- mers, muramyl dipeptide, PGN pentapeptide, GlcNAc, teichoic acid, protein A, and gelatin. The cell surface location of the 70-kDa PGN-binding protein was in- dicated by the ability of PGN to bind to this protein in intact metabolically inactive cells (at 4 “C and in the presence of 0.1% NaN3) and by the ability to extract the 70-kDa PGN-binding protein from viable B lym- phocytes by noncytotoxic concentration of n-octyl-8- D-ghcopyranoside.

Peptidoglycan (PGN)’ is the major cell wall constituent of all bacteria (1). I n viuo, PGN can reproduce most of the major signs and symptoms associated with bacterial infections (fe- ver, inflammation, leukocytosis, abscess formation, macro- phage and lymphocyte activation, arthritis, acute-phase re- sponse, sleepiness, and malaise, etc.) (reviewed in Refs. 2 and 3 ) . Some of these effects may be due to the PGN-induced release of cytokines from macrophages and other cells, whereas other effects may be due to the direct action of PGN on various PGN-target cells (2 , 3). I n vitro, PGN is a strong macrophage and polyclonal B cell activator in all mammals tested (rodents, humans, etc.) (2, 3) , and it even activates invertebrate (e.g. insect) cells (4).

Despite this extensive knowledge of various biologic and immunomodulatory effects of PGN ( 2 , 3 ) , very little is known about the biochemical mechanism of PGN action on host

* This work was supported by United States Public Health Service Grant AR 36640 from the National Institutes of Health and the Project Development Program, Research and Sponsored programs, Indiana University a t Indianapolis. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: PGN, peptidoglycan; ASD, 2-p-azi- dosalicylamido-1,3’-dithiopropionate; two-dimensional PAGE, two- dimensional PAGE with IEF in the first dimension and slab sodium dodecyl sulfate-PAGE in the second dimension; MDP, muramyl dipeptide (MurNAc-L-Ala-o-Glu); PAGE, polyacrylamide gel electro- phoresis; sPGN, soluble PGN.

cells. Using mouse lymphocytes as model PGN target cells and rosetting or [lZ5I]PGN binding assays, we have identified specific PGN-binding sites on these cells (5). Isolation and biochemical characterization of PGN-binding sites is needed as a first step towards providing unequivocal evidence that PGN-induced cell activation is a receptor-mediated process. Identification and characterization of PGN receptors would facilitate studies on the biochemical signal transduction mechanism of cell activation by PGN, and could also enable development of monoclonal antibodies or PGN antagonists that could be useful in preventing PGN-induced clinical man- ifestations associated with bacterial infections.

In this paper we demonstrate the presence of one dominant 70-kDa specific PGN-binding protein in lymphocytes and macrophages, using photoaffinity cross-linking followed by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis (2-dimensional PAGE).

EXPERIMENTAL PROCEDURES AND RESULT$

Demonstration of Specific PGN-binding Sites on B Lympho- cytes-Photoaffinity cross-linking of [‘251]ASD-sPGN or son- icated high M, [’251]ASD-PGN to mouse B lymphocytes, followed by 2-dimensional PAGE revealed the presence of one major 70-kDa protein preferentially binding both of these preparations (Fig. 3, C and E ) . The average PI of this 70-kDa protein was 6.4-6.6 (determined in several other experiments using pH 5-7 ampholites, not shown). This photoaffinity cross-linking represented specific binding, because it was to- tally inhibitable by 400-725 times excess of unlabeled sPGN (Fig. 30 and Fig. 5, below). The photoaffinity cross-linking of sonicated high M, PGN was only partially inhibited by 900 times excess of the same unlabeled PGN preparation (by 50%, as measured by densitometric analysis of autoradiographs, Fig. 3E versus F) , but it was totally inhibited by unlabeled sPGN (not shown). Because these unlabeled PGN prepara- tions did not contain the ASD group, it is concluded that the specificity of [lZ5]ASD-PGN binding demonstrated by cross- linking experiments is for PGN itself, (and not for ASD). These results directly demonstrate the presence of specific PGN-binding sites in B lymphocytes. Two to five PGN- binding spots of similar M , and slightly different PI were usually detected. This PGN-binding site microheterogeneity could represent an experimental artifact (e.g. different num- ber of cross-linker molecules bound per each binding site), different degrees of post-translational modification (phos- phorylation, acetylation, myrystilation, methylation, etc.), or

Portions of this paper (including “Experimental Procedures,” part of “Results,” Figs. 1, 2, 6, and 8, and Table I) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

4713

4714 Peptidoglycan-binding Sites on Lymphocytes and Macrophages

A "~" "

MI

116. 97-

66.

45-

29-

B

45-

29-

I

5 6 7

C E

I

5 6 pH

FIG. 3. PGN-binding proteins in B lymphocytes demonstrated by photoaffinity cross-linking and two-dimensional PAGE. 10' mouse R lymphocytes were incubated with 0.67 pg (24 X IO6 cpm) of [""IIASD- sPGN ( A - D ) or 1.1 pg (21 X 10" cpm) of sonicated high M, ["'II]ASD-PGN ( E and F) for 30 min a t 37 "C in the dark, photocross-linked with UV light, washed, solubilized, reduced with dithiothreitol, subjected to two-dimen- sional PAGE, stained with Coomassie Blue, and dried (shown only for one gel, A ), autoradiographed, rehydrated, and stained with silver stain (shown only for one gel, R) . For cells shown in Q and F, 490 pg of unlabeled sPGN (D) or 1000 pg of unlabeled sonicated high M, PGN (F) were also included in the incubation mixture. Arrows indicate the location of the 70-kDa PGN-binding proteins. Positions of molecular weight standards (kDa) are shown on the left. A, Coomassie Blue-stained two-dimensional PAGE gel. R, silver-stained rehydrated gel shown in A and C. C, autoradiograph of the gel shown in A (proteins binding ['*"I]ASD-sPGN). D, autoradiograph of a gel showing complete inhibition of cross-linking of [""IIASD-sPGN by unlabeled sPGN. E, autoradiograph of a gel showing cross-linking of sonicated high M, [""IIASD-PGN. F, autoradiograph of a gel showing a partial inhibition of cross-linking of sonicated high M, [""IIASD-PGN by unlabeled high M, PGN.

alternate splicing of pre-mRNA. Further experiments will be needed to distinguish between these possibilities. The 70-kDa PGN-binding protein constituted a minor cellular component, it could only be identified by autoradiography and could not be seen even on silver-stained gels, able to detect more than 2000 cellular proteins (Fig. 3R). However, this inability to stain the 70-kDa protein with silver could have also been caused by glycosylation (the possibility of glycosylation of p70 has not yet been investigated, although relatively narrow bands on electrophoresis and similar M , in various cell types and tumor cell lines would indicate either very uniform gly- cosylation or no extensive glycosylation of this protein).

The use of higher concentrations of ["'I]ASD-sPGN and B cells for the cross-linking and longer exposure of autoradi- ographs revealed the presence of other minor PGN-binding proteins (Fig. 4). In addition to the 70-kDa protein, 68-, 46-, 44-, 37-, 34-, 29-, and 27-kDa PGN-binding proteins were detected. However, binding of PGN to these lower M , proteins was weaker than binding to the 70-kDa PI 6.5 protein by several orders of magnitude, as estimated from the density of the spots on autoradiographs. Therefore, the 70-kDa species represents the dominant PGN-binding protein.

Characterization of PGN Binding to B Lymphocytes-Cross- linking of ['251]ASD-sPGN to B cells was competitively inhib- ited by unlabeled sPGN and high M , PGN in a dose-depend- ent manner, as determined by densitometric analyses of au- toradiographs of 2-dimensional PAGE gels (Fig. 5). The ICso

A 6

205- 116- 97- 66-

45-

29-

; A ; ; A ; pH FIG. 4. Demonstration of minor PGN-binding proteins in B

lymphocytes. 2.3 X 10' mouse R lymphocytes were incubated with 1.8 pg (66 X 10' cpm) of ['"'I]ASD-sPGN, photoaffinity cross-linked, and subjected to two-dimensional PAGE and autoradiography as described in the legend to Fig. 3. The 70-, 68-, 46-, 44-, 37-, 29-, and 27-kDa PGN-binding proteins are indicated by arrows. A , Coomassie Blue-stained gel; H, autoradiograph of the gel shown in A.

for sPGN (concentration of unlabeled sPGN needed for 50% inhibition of ['"IIASD-sPGN cross-linking) was 48 pg/ml (calculated by exponential regression analysis), which equals 0.38 pM, assuming average M , = 125,000 (with the range of 0.24-0.95 p~ for the 50,000~200,000 range of M J . The steep- ness of the sPGN inhibition curve slope was normal (i.e. the ratio of the sPGN concentration yielding 90% inhibition to the sPGN concentration yielding 10% inhibition was approx-

Peptidoglycan-binding Sites on Lymphocytes and Macrophages 4715

1102 Inhibitors in order of

potency of inhibitbn (IC50)

"e GlcNPc - MDP - Penlapeptide - ProteinA -c non.0-Ac-2 - TA - non-0-Ac-I -

,_,,___I ,_,__"

-c anhydro-1

-C 0-Ac-I (521 pM) - tri-GlcNAc (10 mM) - HMr PGN (0.80 pM) - sPGN (0.38pM)

r -Q- DxS (124pM)

3 10

0 10' lo2 lo3 l o 4 lo5 lo6 10'

Concentration of inhibitors fnMl

FIG. 5. Competitive inhibition of cross-linkingof ['2"I]ASD- sPGN to the 70-kDa PGN-binding protein on B cells. B cells were incubated as in Fig. 3 with 24 nM ["'I] ASD-sPGN in the medium alone (control) or in the presence of various concentrations of the following inhibitors: MDP, GlcNAc, S. aureu.7 PGN pentapeptide, S. aureu.9 protein A, S. aureu.7 teichoic acid (TA), N . gonorrhoeae non- 0-acetylated peptide-linked PGN dimers (non-0-Ac-Z), non-0-acet- ylated PGN monomers (non-0-Ac-I), anhydro-PGN monomer (an- hydro-I), 0-acetylated PGN monomers (0-Ac-I), tri-GlcNAc, dex- tran sulfate ( D x S ) , S. aureus sonicated high M, PGN ( H M r P G N ) , or S. aurew sPGN. The cells were then subjected to photocross- linking and two-dimensional PAGE as described in the legend to Fig. 3. Inhibition was measured by densitometric analysis of the 70-kDa PGN-binding protein on autoradiographs; the results are means of two to three experiments.

imately 81), indicating simple (via mass action law) interac- tion of a single ligand with a single population of binding sites (24).

Cross-linking of ['ZsI]ASD-sPGN to B cells was partially inhibited (Fig. 5) by Neisseria gonorrhoeae 0-acetylated PGN monomers (IC5o = 469 pg/ml, 521 p ~ ) , by (GlcNAc)3 (ICs0 = 6.6 mg/ml, 10 mM), and also by dextran sulfate (ICs0 = 1024 pg/ml, 124 p ~ ) , which is another B cell mitogen known to bind to lymphocytes (25, 26). The cross-linking was not inhibited by N. gonorrhoeae anhydro-monomer, non-0-acet- ylated PGN monomers, and peptide-linked dimers, muramyl dipeptide, PGN pentapeptide, and GlcNAc. It was also not inhibited by teichoic acid and protein A, which are two other major constituents of the Staphylococcus aureus cell wall, and by unrelated proteins (e.& gelatin).

Because cross-linking experiments were done in a serum- free medium with cells that were handled in RPMI-1640 or Hanks' balanced salt solution without serum and extensively washed and preincubated in a serum-free medium for a t least 1 h a t 37 "C, it may be concluded that immunoglobulins, complement, or other serum components (which may bind to PGN) are not involved in the binding and cross-linking of PGN to the cells. This indicates that PGN binds to the cells by itself, rather than through binding of complexes of PGN with a serum component (e.g. anti-PGN antibodies or com- plement to the Fc or complement receptors) or through bind- ing to a serum component absorbed on the cell surface.

Equally effective PGN cross-linking was also observed at 4 "C in the presence of 0.1% NaN3 (Fig. 6) indicating that metabolically active cells were not needed for the binding of PGN to cells. These results also indicate that PGN binds to the surface of the cells and that the binding is not due to the uptake of PGN by the cells, because low temperature and azide inhibit endocytosis. To further support this conclusion, we have used a non-ionic detergent, n-octyl-8-D-glucopyran- oside, to noncytotoxically release membrane proteins from intact B lymphocytes. This procedure was previously success- ful in releasing cell surface receptors for polyoma virus (27)

A B

Mr 116- 97-

66- /

45- - .

29-

h k ; 4 A ;pH FIG. 7. Two-dimensional PAGE showing n-OCtyl-@-D-glU-

copyranoside-extracted sPGN-binding proteins. H cells were photoaffinity labeled with ['"I]ASD-sPGN, washed, and incubated in 0.1% n-octyl-8-D-glucopyranoside (noncytotoxic concentration); the supernatant was dialyzed, lyophilized, dissolved, reduced, and subjected to two-dimensional PAGE and autoradiography (equivalent of 1.6 X 10" cells). A, silver stained gel; R, autoradiograph.

or lipopolysaccharide (22) without lysing or killing the cells. The major 70-kDa PGN-binding proteins were easily detect- able in n-octyl-D-glucopyranoside extracts of viable B cells (Fig. 7). This result further confirms the cell surface localiza- tion of PGN-binding proteins.

Demonstration of Specific PGN-binding Sites on Mouse B and T Lymphoid and Macrophage Cell Lines-Similar 70-kDa 6.5 PI-specific PGN-binding proteins were also demonstrated in mouse B and T cell lymphomas, T-helper cell clone, and a macrophage cell line, but not in mouse and human erythro- cytes (see the Miniprint section for details).

DISCUSSION

Our results demonstrate the presence of one dominant 70- kDa 6.5 PI PGN-binding protein on the surface of B and T lymphocytes and macrophages, but not erythrocytes. PGN cross-linking to this protein is specific for polymeric PGN, because it is competitively inhibited by sPGN and high M, PGN from two different bacterial strains, but is very weakly inhibited by low M, monomeric PGN fragments or (GlcNAc)n, and is not inhibited by MDP, GlcNAc, PGN-pentapeptide, other staphylococcal cell wall components, or unrelated prod- ucts. This specificity of ['"I]ASD-PGN cross-linking to the 70-kDa protein is similar to the previously determined speci- ficity of ["'IIPGN binding to lymphocytes in a binding assay (5). Also, the ICso for sPGN obtained here in competitive inhibition of cross-linking (0.24-0.95 p ~ ) is similar to the ICs0 for high M, PGN (0.12-0.46 p ~ ) previously obtained in competitive inhibition in the binding assay (5).

The presence of PGN-binding proteins on both B and T lymphocytes is not surprising. Although PGN is mitogenic for only B lymphocytes in mice (10, ll), PGN is mitogenic for both B and T lymphocytes in humans (28). Moreover, using ['2sI]PGN binding assay (5), recently we have also detected specific binding of PGN to both B and T mouse lymphocytes:' Similarly, the presence of lipopolysaccharide- binding sites was detected on both T and B lymphocytes, although lipopolysaccharide is only a B cell mitogen (19). The presence of PGN-binding sites on T lymphocytes suggests that PGN, like several other T cell activators (29), in some species may not be a mitogen by itself, but it may still function as an activator or a co-mitogen in conjunction with other

Several pieces of indirect evidence suggest that this PGN- binding site may serve as a functional lymphocyte-activating PGN-receptor: (a) this binding site is located on the cell

ctimnl;

R. Dziarski, unpublished data.

4716 Peptidoglycan-binding Sites on Lymphocytes and Macrophages

surface of PGN target cells and is specific for PGN; ( b ) the concentrations of PGN needed for lymphocyte activation (10, 11) are similar to the ICbo of PGN obtained by two independ- ent techniques (inhibition of cross-linking and binding assay) (5); (c) PGN is able to directly activate highly purified lym- phocytes in conventional or serum-free media (which makes it unlikely that lymphocytes are activated by products of other cells or by possible complexes of PGN with serum compo- nents) (10, 11, 15, 16); ( d ) there is an excellent correlation between the ability of different PGN preparations to stimulate lymphocytes, to bind to lymphocytes, and to inhibit binding or cross-linking of radiolabeled PGN to lymphocytes (5, 10, 11, 15); and ( e ) a pertussis toxin-sensitive G protein is in- volved in PGN-induced B cell activation (and processes which involve signaling through G proteins are typically receptor- mediated mechanisms) (16). However, to provide unequivocal direct evidence that PGN-induced cell activation is a receptor- mediated process, these receptors will have to be first isolated and biochemically characterized, then their amino acid and cDNA sequences will have to be established, and then unre- sponsive cells will have to be converted into responsive cells by reconstitution with purified PGN-binding sites or by in- sertion of the genes coding for PGN-binding sites. The results presented in this paper provide a first step towards these goals.

The 70-kDa PGN-binding protein is a minor cell surface component which is also consistent with the proposed recep- tor function of this molecule. This PGN binding site is most likely different from the MDP-binding sites (30, 31) (which are also specific for serotonin (32)), because MDP does not inhibit PGN binding and because the affinity of MDP binding to its binding site is 2-3 orders of magnitude higher than the affinity of PGN binding (5, 30). The relationship of the 70- kDa PGN-binding protein to other known lymphocyte and macrophage cell surface molecules, such as cytokine and growth factor receptors or CD antigens, is currently unknown. However, we have obtained strong evidence, presented in our companion paper (33), that this 70-kDa PGN-binding protein is identical with the 80-kDa lipopolysaccharide-binding pro- tein discovered by Lei and Morrison (19, 22). These results suggest that this 70-kDa protein binds to the polymeric (GlcNAc-MurNAc), of PGN and (GlcNAc)2 of lipid A (33).

Acknowledgments-I am grateful to Michele Hadhazy for excellent technical assistance; Dr. A. Zeiger for his generous gift of the S. aureus strain and helpful advice on purifying sPGN Drs. R. S. Rosenthal, J. A. Rupley, M. Parant, R. Scartazzini, and K. Scheibli for their generous gifts of reagents; Dr. L. M. Switalski for amino acid analyses; Dr. 0. Spivey for his advice on photoaffinity cross- linking; and Drs. W. M. Anderson and T. J. Mueller for helpful discussions and use of their equipment.

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477

2. Heymer, B., Seidl, P. H., and Schleifer, K. H. (1985) in Immu- nology of the Bacterial Cell Envelope (Stewart-Tull, D. E. S., and Davies, M., eds) pp. 11-46, J. Wiley & Sons, New York

3. Dziarski, R. (1986) in Biological Properties of Peptidoglycan (Seidl, H. P., and Schleifer, K. H., eds) pp. 229-247, Walter De Gruyter, Berlin

4. Kanost, M. R., Dai, W., and Dunn, P. E. (1988) Arch. Insect Biochem. Physiol. 8, 147-164

5. Dziarski, R. (1987) Cell. Immunol. 109, 231-245 6. Zeiger, A. R., Wong, W., Chatterjee, A. N., Young, F. E., and

7. DePedro, M. A., and Schwarz, U. (1980) F E M S Microbiol. Lett.

8. Perkins, H. R. (1969) Biochem. J . 111, 195-205 9. Dziarski, R. (1984) Immunology 53,563-574

Tuazon, C. U. (1982) Infect. Immun. 37, 1112-1118

9, 215-217

10. Dziarski, R., and Dziarski, A. (1979) Infect. Zmmun. 23,706-710 11. Dziarski, R. (1980) J. Immunol. 125, 2478-2483 12. Dziarski, R., and Kwarecki, K. (1976) Zentralbl Bakteriol Micro-

13. Martin, S . A., Rosenthal, R. S., and Biemann, K. (1987) J. Biol.

14. Rupley, J . A. (1964) Biochim. Biophys. Acta 83,245-255 15. Dziarski, R. (1988) Cell. Immunol. 111, 10-27 16. Dziarski, R. (1989) Eur. J. Immunol. 1 9 , 125-130 17. Ruoho, A. E., Rashidbaigi, A., and Roeder, P. E. (1984) in

Membranes, Detergents, and Receptor Solubilization (Venter, J. C., and Harrison, L. C., eds) pp. 119-160, Alan R. Liss, New York

18. Wollenweber, H.-W., and Morrison, D. C. (1985) J. Biol. Chem.

19. Lei, M.-G., and Morrison, D. C. (1988) J. Zmmunol. 141, 996-

20. O’Farrell, P. H. (1975) J. Biol. C h m . 250,4007-4021 21. Roberts, M. R., Baumbach, G. A., Buchi, W. C., Denny, J. B.,

Fitzgerald, L. A., Babelyn, S. F., and Horst, M. N. (1984) in Molecular and Chemical Characterization of Membrane Recep- tors (Venter, J. C., and Harrison, L. C., eds) pp. 61-113, Alan R. Liss, New York

22. Lei, M.-G., and Morrison, D. C. (1988) J. Zmmunol. 141, 1006- 1011

23. Switzer, R. C., Merril, C. R., and Shifrin, S. (1979) Anal. Biochem. 98,231-237

24. Limbird, L. E. (1986) in Cell Surface Receptors: a Short Course on Theory and Methods, pp. 51-96, Martinus Nijhoff Publish- ing, Boston, MA

25. Parish, C. R., and Snowden, J. M. (1985) Cell. Immunol. 91,201- 214

26. Parish, C. R., McPuhn, V., and Warren, H. S. (1988) J. Immunol.

27. Marriott. S. J.. Griffith, G . R., and Consigli, R. A. (1987) J. Virol.

biol. Hyg. Abt. I Orig. A S5, 393-406

Chem. 262,7514-7526

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28. Levinson, A. I., Dziarski, A., Zweiman, B., and Dziarski, R. (1983)

29. Weiss, A. (1989) in Fundamental Immunology (Paul, W. E., ed) 2nd Ed, pp. 359-384, Raven Press, New York

30. Silverman, D. H. S., Krueger, J. M., andKarnovsky, M. L. (1986) J. Immunol. 136,2195-2201

31. Tenu, J.-P., Adam, A., Souvannavong, V., Yapo, A., Petit, J.-F., and Douglas, K. (1989) Int . J . Immunopharmacol. 11,653-661

32. Silverman, D. H. S., Wu, H., and Karnovsky, M. L. (1985) Biochem. Biophys. Res. Commun. 131, 1160-1167

33. Dziarskl, R. (1991) J . Biol. Chem. 266,4719-4725

Peptidoglycan-binding Sites on Lymphocytes and Macrophages Supplementary Material

t o

Demonstration 01 Peptldoglycan Binding Sites on Lymphocytes and Macrophages by Photoafflnlty Crosslinking

b y

Roman Dzlarskl

4717

EXPERIMENTAL PROCEDURES

Pepfldoglycan (PGN). Soluble PGN (sPGN) that is secreted by Sfaphylococcus aureus In the presence of penicillin (6). was obtained as described (6) wlth some modifications (7). S. aureus (strain Rb kmdly provided by Dr. A. Zeger. Thomas Jefferson University) was grown for 60 min in the synthetic (dtalyzable) minimal cell wall medium supplemented with [14C]Ala and penicilltn G: the cullure supernatant was concentfaled. dialyzed and fractionated on a Sephadex G-100 column (6). The peak of

column. which specifically binds D-Ala-D-Ala of uncrosslinked PGN (6-8). The column [14C]Ala-labeled material was collected and applled Onto vancomycin-Sepharose

was washed with PES. pH 7.2. and sPGN was eluted with a gradient of pH 8.0-10.2 of NH4OH m H20 (6.7). The only material that eluted from lhe column was a closely spaced double peak (at pH 8.4-8.7) 01 [l4C]Ala-Iabeled material with a maximum absorbance at 218 nm. The fractions conlatnmg [l4C1Ala were pooled. concentrated. and dialyzed. All these fractions contained sPGN that were undistingulshable by chemlcal analyses, PAGE, bmding (crosslinking). lnhlbition 01 bmdmg. and B cell

analyzer (9). revealed the presence of (nmollmg): QluCOSamlnelmuramiC acld (1971). mllogenlcity assays. Sugar and amino acld composil~on. determined on an amino acfd

Gly (4927). Ala (3988). Glx (1414). Lys (1367). Nle (416). Ser (333). Phe (162). Pro (68). ASX (58). Leu (43). Thr (22). Val (20). and 118 (17). which indicates that 95% of ammo acids .n this preparation were typical for S. aureus sPGN. the remaining 5% were allernatwe amino acids that can substilule the lypical amino acids, whereas there was no detectable contamination with aromatc and sulphur.containing amino acids, whch are never found in PGN (1). The concentrations of sPGN were determined on an ammo acld analyzer and were In excellent agreement with the concentrat~ons determined spectrophotometrically (OD218) using olher PGN preparations as standards.

Sonicated high M, PGN was obtained from insoluble S. aureus PGN by sonication as described (5) and chemically analyzed on an amino acid analyzer and tested for contaminations as before (5.9-12). The MI 01 this sonicated PGN is 2-8 x 106, as determmed by gel fillration of [r251]PGN (5). It gives one band (that stays on top of the gel) m overloaded (100 pgllane) SDS slab PAGE when statned with Coomassie blue (5) . This band disappears lollowing PGN ddgestlon with lysozyme. lysostaphin. or N-acetyl- muramldase SG. but not wlth trypsm. pronase, RNase. or DNase (5).

kindly provided by Dr. R.S. Rosenlhal (Indiana University. Indianapolis. IN): non-0- The lollowing HPLC-purilied Neisserta gonorrhoeae PGN fragments (13) were

acetylated monomer ( G ~ C N A C - M U ~ N A C - L - A ~ ~ - D - G I U - A Z ~ ~ - D - A ~ ~ ) . 6-0-acetylated monomer. (1.6-anhydr0)MurNAc-monomer (approx. M, 01 monomers was 900). and pepttde-crosslinked “on-0-acetylated dimer (GlcNAc-MurNAc-L-Ala-D-Glu-A2pm.[D. Ala-D-Ala]-A2pm-D-Glu-L-Ala”urNAc-GlcNAc. approx. M, - 1800).

(muramyl dipeptide. MDP. MI - 493) (kmdly donated by Dr. M. Parant lrom CNRS. Paris. Other resgenfs. As synthetic analogs of PGN Iragments. MurNAc-L-Ala-D.Glu

and by Drs. R. Scanazzini and K. Schelbly from Clba-Geigy. Easel) and staphylococcal

MO) were used. S. aureus ribitol leichoic acid (approx. Mr I 4400) was purified as PGN pentapeptide (L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. MI - 488. from Sigma, St. Louis,

before (12) and protein A (M, - 42,000) was obtained from Pharmacia Fme Chemicals (Piscataway. NJ). Chitolriose [(GIcNAc)~. MI - 660) was kindly donated by Dr. J A.

obtalned by phenol-water extractlon method. approx. average M, - 15,000) was from Rupley from the University of Arizona. Tucson (14). Purified hpopolysaccharrde (LPS.

Escherichia COB 0113 (from Rlbl lmmunochem Research, Hammlton. MT) or 0127.88 (from Sigma). N-acetyl-muramidare SG was from Dainippon Pharmaceutcal Co. (Tokyo, Japan). GIcNAc. dextran sulfate ( M , - 8,000). lysozyme (from chicken egg white). lysostaphtn (from Slaphylococcus sfaphylolyfcus). pronase (nonspecific protease type

pancreas). and all other reagents were from Slgma. unless otherwse mdlcated. XXI from Sfrepromyces griseus). trypsm. RNase A and DNase I (all three from bovme

Cells. Splenic B lymphocytes from female BALBlc mice (6-13 wk old from Harlan Sprague-Dawley. Indianapolis. IN) were obtamed as before (15.16). following injection with goat anti-mouse thymocyte serum and in vifro treatment wlth mOnOclonat IgM anti.Thyl.2 and complement. Adherent cells were removed by a passage through glass wool column (15). The B cells were analyzed for purity as belore (15.16): they typically contamed >90% surlace Ig-positive cells by

A (stimulation indexes at) (15.16). The following cell lines or clones (obtained from ~mmunofluorescence. 4 % macrophages. and were totally unresponsive to PHA and Con

was adapted to 2% Serum) were also used: A20 (BALWc mouse B cell lymphoma), YAC-1 ATCC and malntatned in culture as recommended by ATCC. except for P388D1. whch

clone). and P388D1 (DEN2 mouse macrophage cell Ilne). Mouse (BALBlc) and human (NSn mouse T cell lymphoma). D10.G4.1 (AKR mouse conalbumin-SpecofIc T-helper cell

erylhrocytes obtained from heparinnzed blood were also used.

itasllASD derlvatlzatlon 01 PGN. SASD (sulfosuccinimidyl 2-p-azido- salicylamtdo-1.3”dithiopropionate. 2 x 400 pg. from Pierce Chemical Co.. Rockford. IL. dissolved at lOOx concentration in DMSO) was reacted with 840 pg sPGN or 1 mg Sonicated high M, PGN In 0.5 ml 01 0.1 M borate buffer. pH 8.5. hwce for 30 min at 22% (17.18). The reaction mixture was termmated by adding 0.1 M arginine for additional

photoactivatton) was performed in the dark or with red light source. ASD (2-p-azido- 10 mm. centrifuged at 2000 x g lo remove aggregates and dialyzed. All handling (until

salicylamido-l.3”dithiopropionate)-PGN (84-100 pg) was then iodinated wilh carrier- free Na[1251] (1-2 mCI from New England Nuclear, Boston. MA) by the chloramine-T method (18). and unbound [1251] was separated from [l25I]ASD-PGN by gel liltration on Sephadex G-25 (5.18). The ranges of specific activities 01 [ ~ ~ ~ I J A S D - S P G N and high MI PGN were 7.28 pCilpg and 8-15 pcilpg. respectively. Tests showtng that the label was indeed attached lo the derivatized PGN are described below in RESULTS.

Pholoafllnlfy crossllnklng of [‘ZslIASD-PGN lo cells. The procedure of Lei and Morrison (19). developed for photoaflinily crosslinking of [125I]ASD-LPS to lymphocyles was used. Cells (10 x 106 in 0.22 ml Hanks BSS with 0.05% gelatin) were incubated with [~~SIIASD-SPGN (typically 0.67 pg) or [l251]ASD-PGN (1.1-1.2 pg) for 30 min at 37OC in the dark, then exposed to UV light lor 10 min et 22OC. washed three times by centrifugation. solubdized by ullrasonication in 9.2 M urea with 5 mM K2CO3. 2% Pharmalyle, 0.4 mM phenylmethyl-sulfonyl fluoride. 2% Nonidet P-40 and 0.25

mglml DTT. Cell lysates were heated tor 5 min at 50% (to reduce ASD’s disullide bonds) and subjected to 2D.PAGE (IEF and SDS-PAGE). Prellminary experiments on the kinetics 01 bmding have established that binding of [*25l]ASD-sPGN. similarly lo [lPSI]PGN binding (5). was completed in 15.30 min at 370C. In some experiments. incubation 01 cells with [ ~ ~ ~ I I A S D - S P G N and crosslinking was done at 4% andlor in the presence of 0.1% NaN3. as tndicated in RESULTS. It should be noted that because the iodmatable groups and the photoactivatable azido groups are distal to the reducible dtsulfide bonds, the reduction after crosslinkinp transfers the label from [l25I]ASD- PGN to the PGN-binding sites and elimmates PGN (which would hinder subsequent electrophoretic analysts). AS a control lor specilc PGN crosslinking. smlar procedure was performed with cells incubated as above plus excess of unlabeled sPGN.

experiments. the inhibitors were pre-mbxed with the labeled PGN before addtlton to the PGN. or other products. as indicated in RESULTS. In these competitive inhibktlon

cells.

2D-PAGE was performed with isoelectric locusing (IEF) on 1.5 x 120 mm pels with 2% Two dlmenslonal slab polyacrylamide gel elecfrophoresls (2D-PAGE).

Pharmalyte (291. pH 3-10. or 1% pH 3-10 plus 1% pH 5-7. or in some experiments 2% pH 5-7: from Pharmacia Fme Chemicals) in the first dimension (with samples applied at the basc end). and 11% SDS-PAGE in the second dimension (19-21). The gels were stained with Coomassie blue, dried and autoradiographed (typically lor 2-5 days) uslng Kodak XAR-5 f h and mlensifying screens a1 -80% (19.21). Spot density was determined usmg a gray scale scanner (MSF 300GS. Microtek. Torrance. CA) and Biosoft (Cambridge, UK) Scan Analyss software. More Sensltlve Silver stain (Rapid-Ag-Staln, ICN. Cleveland. OH) was used on occasions. but because it quenches autoradiography (21). it was performed on rehydrated gels after autoradlography was completed.

Other fechnlques. Enzyme digestions (lysozyme. lysostaphin. muramidase SG. pronase. trypsm. RNase, or DNase. at 0.1 mglml) of PGN preparations were performed at 37% lor 18 hrs. unless otherwise indicated. PGN labeling with [1251]Bolton.Hunter reagent was done as before (5). 2.5.diphenyloxazole (PP0)-enhanced fluoroaulo. radlography was performed as described (21). For extract&on of cell surface PGN- bmdmg proteins with non-cytotoxic concentralion of n-octyl-R-D-glucopyranoside (OG) (22). 2 x 108 B cells were photoaffinity labeled with [l251]ASD.sPGN. washed.

g. The supernatant was dialyzed against H20. lyophilized. dissolved in solubilmng- incubated in 4 ml of 0.1% OG in Hanks BSS for 30 min at 22% and centrifuged at 300 x

reduclng buffer, and subjected to 2D-PAGE. In parallel Control experiments. the viabiltty of B cells measured by trypan blue exclusion belore and alter simllar

([JH]thymidine incorporation) was performed as before (15.16). Limulus lysate assay incuballon with up to 0.15% 00 remained 198%. B cell mitogen~ctty assay

(Eloxate. Sigma) was performed as recommended by S V a .

RESULTS

Charsclerlzarlon 01 SPGN. PasI]ASD-sPGN snd hlgh M, P2sI]ASD-PGN. The purity of sPGN was established by the following criteria: (a) Sugar and ammo acid cOmpOSition revealed no detectable contamination with sugars or aromatic or sulphur. contaming amino acids that are never found in PGN (1). (b) There was no evidence of contamlnating protem bands in sPGN preparations on overloaded (150 pgnane) sdver- stamed PAGE gels, able to detect 0.3 ng 01 protein per band (21.23) (Fig, 1 A note that

of sPGN endogenously labeled wjth [“CIAla revealed the presence 01 one uniform broad hlgh MI sPGN stalns very weakly with silver). (c) lluoroautoradiography of PAGE gels

band. characteristic of a polysaccharide with M, ranging from 50.000 to 200,000 (Fig. 18). This band disappeared following sPGN dlgestlon wbth lysozyme or lysostaphin. but not with trypsm. RNase, or DNase (no1 shown). (d) sPGN was also free from any signilicant accidental endotoxm contamination (<lo ng endotoxinlmg sPGN. as determined by Llmulus Lysate assay).

A B C

205-

205- I 2 0 5 - m 116- 97- ”

D

29- 29- 29- 0 - - - d

1 2 3 4 5 6 7 8 2 3 4 5 6 7 8

Fig. 1. SDS-PAGE (515%) of sPGN and aenaltlvlly to enzyme dlgestlons. A. Silver Stained gel of sPGN (150 pgllane); note only weak staining of high Mr sPGN and no contamination with proteins. 6. Fluoroautoradiography (PPO-enhanced) showing sPGN (150 pgllane) endogenously labeled with [I‘CIAla. C. (L D. Coomassle blue stain

cpmllane) with enzymes (0.1 mglml. 20 plllane): lane 1. MI standards: fane 2. (C) and autoradiograph (D) showing digestion of [ ~ ~ ~ I I A S D - S P G N (13 pg - 40.000

undigested (no enzymes): lane 3. lysozyme: fane 4. lysostaphin: lane 5. pronase: lane 6. trypsin: l tne 7. RNase: lane 8. DNase. Posltions of molecular wetght Standards (kDa) are shown on the left.

4718 Peptidoglycan-binding Sites on Lymphocytes and Macrophages

times more actlve on per weight basis. but less active on per mol basis than sonicated sPGN was mitogenic tor mouse B lymphcqtes (Table I). sPGN was live to ten

high MI PGN (assuming average MI- 125.000 lor sPGN and 5.000.000 lor hgh M, PGN). However. because 01 limited accuracy 01 MI determination 01 sPGN by electrophoresls and hrgh MI PGN by gel filtration. and unknown extent 01 aggregataon of these preparations in solution, the mitogenic ptency 01 these preparations can be considered approximately equivalent on per mol basis.

lines and hipher cnncentrations 01 [~~~I IASD-SPGN (not shown). PGN-bindinp proteins could not be detected on mouse and human erythrocytes. even upon extensive overexposure 01 autoradiographs (lour times longer than tor other cells).

A B C I

Mlrogenlclty of sPGN and sonicated high M, PGN for mouse B lymphocytes TABLE I

-~ Preparation Concentration incorporation. index

[3H]thymidine Stimulation

I / /

; A i i . ; ; ; A i pH

[125i]ASD-sPGN to mouse B lymphocylea. B cells were incubated with [f25t]ASD- Ftg. 6. E l e c t of temperature and azide on photoalllnily croaslinklng 01

described in Rg. 3. but the 90 min incubation and 10 min crosslinking were done at 4% sPGN. photoalfinity crosslinked and subjected to 2D.PAGE and autoradiography as

autoradlograph 01 the gel shown in A. C. autoradiograph of a parallel control incubated in the presence 01 0.1 X NaN3 (AB). A. Coomassie blue stained 2D-PAGE gel; 8 .

tor 30 min at 37% and crosslmked lor 10 min at 22%.

A. I B C Mr

-10-

80-

07-

45-

¶e-

20 (160) 4 (32)

100 (800)

5 H I

889 t 25 566 t 20

1609 +. 15

206*3 374 t 59 779 t 23

1616 f 33

3.4 5.3 9.6

1.2 2.2 4.6 9.6

High Mr PGN

125 (25) 625 (125)

25 i5i

LPS 15 (1000) 3385 t 132 20

PHA 38 (302) 175 * 20 1 .o Con A 3 (29) 205 * 26 1.2 "_ a [JHIthymidine incorporation into B cells cultured tor 2 days with t h e i n d l c x d preparattons was measured. The results are means lrom three Cultures in one representatlve experiment.

"_

sPGN and Sonicated htgh Mr PGN preparattons (and not to Some minor otherwise The following two lines 01 evidence indicate thai ['2JI]ASD was indeed bound to

undetectable contaminant or to a minor subfraction 01 PGN). First. the pattern 01 [1251]ASD-labeled material on SDS-PAGE gels was identical to the pattern 01 tluoroautoradiographed endogenously labeled sPGN (Ftg. 10.0). or ['251]8olton.Hunter- labeled matertal. or Coomassie blue Stained material (tor sonrcated high M, PGN. Fig. 2) Second. the label was solubilized lollowmg digestion with PGN.lytic enzymes (lysozyme. lysostaphin. muramidase SG. and in the case of sPGN also pronase). and was resistant to other enzymes (trypsin. RNase, DNase) (Figs. 1 and 2). Sensitnvity 01

with tree ammo groups (glutamme or lysine). Lack 01 sensitivity to pronase 01 ["WIASD-sPGN to pronase suggests that the label is bound to one 01 the amino acids

sonicated high Mr PGN is due to highly crosslinked nature 01 this PGN. as opposed to uncrosslmked sPGN (t.6). Digestion with lysozyme. lysostaphm and muramidase SG (but not trypsin. RNase. and DNase) removed the abilihl 01 ['~SIIASD-SPGN to crosslink to lymphocytes (not shown). It Should also be noted that treatment 01 [ I ~ ~ I I A S D - S P G N and (I25IIASD-PGN with DTT at 55% totally converted the label into low Mr spectes. mgratlng on SDS-PAGE with the front 01 the buffer (not shown), indicatlng that all [I2511 was bound through a reducible S-S bond to ASD. Similar treatment 01 [1251]0olton-Hunter-PGN did not Solubilize the label.

; ; ; ; A ; pH

Fig. 8. PGN binding proteins in mouae B and T lymphoid end macrophage cell l ines demonstrated by photoalfinity croasllnklng and 20-PAGE. lo7 cells were incubated with 0.67 pg 01 [~~~I IASD-SPGN (A.B.D.E.G.H.J.K) or also with 245 pg 01 unlabeled sPGN (C.F.1.L) and processed as in described in Fg. 3. A.D.G.J. Coomassie blue stamed 2D-PAGE gels: B.C.E.F.H.I.K.L. autoradlographs. A. B. C. A20 0ALBh 8 cell lymphoma.

I) E MI

Ira- 07-

8s-

45-

19.

; ; ; ; a ; Fig. 8. D. E. F. YAGI AJSn T cell lymphoma.

G H

MI

116. 97'

s¶

4 6

2 0

i b ; ; b ;

Fig. 8. G. H. I, D10.G4.1 AKR T-helper cell Clone.

J K M

¶05. 7%.

ed 07

45

19

A b i i b i Fig. 8. J. K. L. P388D1 DEN2 macrophage cell line.

F

C D """

A 0- - B

b ; pH " I

ne-

66-

45-

29-

97-

1 2 3 4 8 6 7

Flg. 2. SDS-PAGE of sonicaled hlgh Mr PGN and senslllvlly to enzyme digestions. A. Autoradiograph 01 5.15% pel showing digestion 01 Sonicated high MI ['25l]ASD-PGN (24 pg - 42.000 cprnllane) with enzymes (0.1 mglml. 20 plllane): lane 1. undigested (no enzymes): lane 2. lysozyme: lane 3. lysostaphin: lane 4. pR)nase; lane 5. trypsln: lane 6. RNase: lane 7. DNase. 8 . C. 8 0. Coomassie blue stain (8) and autoradtographs (C 8 D) of 2.5% acrylamide + 0.5% agarose gels showing digestion 01 sonicated high MI [t251]8oIton.Hunter-PGN (5) (B a C. 105 pg - 48.000 cpmnane: D. 54 pg - 28.000 cprnllane) with lysostaphin (8 8 C): lane 1. undigested (no enzyme); lane 2. 45 min: lane 3. 90 man: lane 4. 180 min; or with muramidase SG (D): lane 1. undigested (no enzyme); lane 2. 22 min. lane 3. 45 min; lane 4. 90 mln: lane 5. 3 hrs; lane 6. 6 hrs: lane 7. 12 hs; lane 8. 24 hrs. Positions 01 molecular Weight standards (kDa) are shown on the left.

lymphoid and macrophage cell Ilnee. Similar 70 kDa 6.5 pl specific PGN-binding Demonstration of speclflc PGN-binding sires on mouse 8 and T

proteins were demonstrated in mouse A20 BALBlc 0 cell lymphoma (FIQ. 8 A.0.C). YAC.

and P388D1 DEN2 macrophage cell line (Fig. 8 J.K.L). The binding was also inhibitable 1 AJSn T cell lymphoma (Fig. 8 D.E.0. D10.G4.1 AKR T-helper cell clone (Fig. 8 G.H.1).

by excess 01 unlabeled sPGN. Five PGN-binding spots of similar Mr and slightly different pl were usually detected. AS already indicated. the reasons lor and the significance 01 this PGN-binding site microheterogeneity is currently not clear. As with normal B cells. few minor PGN-binding proteins were also Seen with some cell

i pH