From the Department of Immunology, School of Microbiological,
Virological, and Immunological Sciences, The Medical School, University
of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne,
Tyne and Wear, NE2 4HH, United Kingdom
We studied endosomal proteolysis of the surface
fibrillar M5 protein from viable Streptococcus pyogenes as
an essential step involved in major histocompatibility complex class
II-restricted antigen processing of two immunodominant CD4+
T-cell epitopes (17-31/Ed and 308-319/Ad).
Intracellular proteolysis of viable streptococci for presentation of
17-31, bound by serine proteinase cleavage sites, was mediated by
serine proteinases, whereas processing of soluble recombinant M5
protein required in addition cysteine proteinases. Furthermore, processing of 17-31 was resistant to ammonium chloride and thus was
not dependent on endosome acidification. Cysteine and serine proteinase
cleavage sites were located adjacent to 308-319, and its processing
was dependent on serine, cysteine, and aspartic proteinases, as well as
on endosomal acidification. The data suggest that antigen
processing of two major T-cell epitopes on streptococcal M5 protein
occurred in different endosomal compartments by different classes
of intracellular proteinases.
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INTRODUCTION |
Exogenous antigens have to be subjected to intracellular enzymatic
processing by professional antigen-presenting cells for major
histocompatibility class II
(MHC-II)1-dependent
presentation of peptide fragments to CD4+ T-cells (1).
Peptides released in late endosomes are transported to specialized
MHC-II binding compartments for recognition by MHC-II molecules (2, 3).
A chaperone molecule (the invariant (Ii) chain) associated with MHC-II
molecules targets the nonameric 
-Ii complex via endoplasmic
reticulum and trans-Golgi network to endosomes. Ii chain is
then sequentially cleaved by aspartic and cysteine proteinases, and
HLA-DM (H2-M in mouse) facilitates the exchange of the class
II-associated invariant chain peptide for processed antigenic peptides
(4). Recent evidence suggests that antigen processing of certain
proteins (influenza hemagglutinin and myelin basic protein) can also
occur in early endosomal compartments with peptides being loaded on
MHC-II molecules recycled from the plasma membrane (5, 6) to
specialized class II vesicles.
Intracellular proteolytic enzymes have been shown to be intrinsically
associated with antigen processing (7). Indeed, at least two out of
four classes of intracellular endopeptidases, namely aspartic and
cysteine proteinases, have been shown to be directly involved in
endosomal proteolysis of model antigens as well as in enzymatic
processing of Ii chain (2, 7-9). Thus, cathepsin S has been reported
to be primarily engaged in degradation of Ii chain, thus facilitating
the downstream peptide loading on MHC-II molecules (10). However, other
classes of intracellular enzymes, i.e. metalloproteinases, may
contribute to the overall efficiency of antigen processing (11). It has
also been shown that the proteolytic activity of endosomal enzymes is
regulated by the pH gradient within the endosomal pathway (7, 12, 13), suggesting that different classes of proteinases may be implicated in
distinct MHC-II antigen processing compartments.
We have previously shown that two
CD4+-dependent T-cell epitopes
(17-31/Ed and 308-319/Ad) located on the
surface fibrillar M5 protein, the main virulence factor and protective
antigen of Streptococcus pyogenes, were efficiently
processed from viable streptococci for MHC-II-restricted presentation
to specific CD4+ T-cell clones and T-cell hybridomas
(14-17). In this report we present evidence that processing of 17-31
was mediated by serine proteinases, whereas 308-319 required serine,
cysteine, and aspartic proteinases, suggesting that two epitopes on the
same protein engage largely different endosomal compartments for
MHC-II-dependent antigen processing.
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EXPERIMENTAL PROCEDURES |
Cells and Chemicals--
The murine macrophage cell line J774A.1
(H-2d, ATCC TIB 67) was used as antigen-presenting cells.
T-cell hybridomas (HX17 and HY2) obtained by polyethylene glycol fusion
of two M5 protein-specific T-cell clones (X17 and Y2) with BW5147
(TCR


) cells (kindly provided by Dr.
P. Marrack, Denver) were specific for epitopes 17-31/Ed
and 308-319/Ad of group A streptococcal type 5 M protein,
respectively, as reported previously (17). Antigen-presenting cells and
T-cell hybridomas were grown in RPMI 1640 medium containing 3.0 mM L-glutamine, 0.05 mM
2-mercaptoethanol, and 10% fetal bovine serum (v/v). S. pyogenes (strain Manfredo) was grown overnight in RPMI 1640 with 10% fetal bovine serum, washed once in phosphate-buffered saline and
the concentration was adjusted spectrophotometrically to 3 × 108 colony-forming units/ml (A600 = 0.6). Culture media, chemicals, and metabolic inhibitors (Table
I) were from Sigma Chemical Co. (Dorset,
UK).
Soluble Recombinant M5 Protein (rM5) and Synthetic
Peptides--
The recombinant M5 protein (rM5) from type 5 S. pyogenes strain Manfredo was cloned and expressed in
Escherichia coli LE392 as described previously (14, 18).
Synthetic peptides covering two T-cell epitopes on the M5 protein of
S. pyogenes were purchased from the University of Newcastle
upon Tyne, Facility for Molecular Biology: (i) 15-33 peptide contained
epitope 17-31/Ed, and (ii) 300-319 peptide covered
epitope 308-319/Ad (14).
Antigen Processing Assay--
Following adherence (6 × 104/well) (48-well plates, Bibby Sterilin Ltd.,
Staffordshire, UK) for 1 h, J774A.1 macrophages were treated with
inhibitors for 30 min, unless stated otherwise. Viable streptococci
(3 × 106/well), rM5 (1.0 µg/ml), or synthetic
peptides (4.0 µg/ml) were added, and plates were incubated at
37 °C in a humidified CO2 incubator for 1 h, after
which nonphagocytosed bacteria were killed with gentamycin (50 µg/ml), and the plates were incubated for an additional 3 h. The
macrophages were then fixed with 1.0% paraformaldehyde for 10 min,
washed with phosphate-buffered saline, and T-cell hybridoma cells were
added (3 × 104/well) for 24 h. The culture
supernatants were removed and stored at
20 °C for subsequent
interleukin-2 assay. To ensure that the inhibition observed resulted
from the specific, rather than nonspecific, effect of inhibitors, the
viability of J774A.1 cells before fixation was confirmed in all
experiments. All experiments were performed at least three times, and
the data for a representative experiment are shown.
The response of T-cell hybridomas was measured as proliferation of
CTLL-2 cells (104/well) in the presence of T-cell hybridoma
culture supernatants in flat-bottomed 96-well microtiter plates (Becton
Dickinson Labware, New Jersey). Each supernatant was tested at a 1:2
dilution in duplicate for 24 h at 37 °C in a humidified
CO2 incubator, followed by pulse-labeling with 0.4 µCi of
[3H]thymidine (TRA310, specific activity 2.0 Ci/mmol;
Amersham International plc, Buckinghamshire, UK) for 18 h. Cells
were harvested on glass fiber membranes, and radioactivity was
quantitated using a direct beta counter (Matrix 9600, Packard
Instrument Company, Meridan, CT).
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RESULTS |
Role of Endosome Acidification in Antigen Processing--
For
antigen processing to take place, both the enzyme and the appropriate
pH should be present within one endosomal compartment of the
antigen-presenting cells to ensure efficient proteolysis. It has been
shown that although intracellular proteinases are present in all
endocytic compartments, to attain full enzymatic activity inactive
proenzymes require exoproteolytic maturation controlled by the pH
gradient within the endosomal pathway (7, 19). Hence, endosomal
acidification plays a pivotal role in antigen processing and
presentation.
To study the importance of endosomal acidification for processing of
the streptococcal M5 protein for presentation of two immunodominant
T-cell epitopes (17-31/Ed and 308-319/Ad) to
specific T-cell hybridomas, we used two specific metabolic inhibitors.
Monensin is a carboxylic (cationic) ionophore (20) that intercalates
into membranes and mediates exchange of protons for potassium ions,
thus effectively raising endosomal pH (12). Ammonium chloride is a weak
base that promotes alkalinization of the endosome content (12).
Ammonium chloride had no apparent effect on presentation of 17-31
(Fig. 1A). In contrast, this
inhibitor blocked processing of 308-319 from viable bacteria or rM5
protein, and reduced presentation of the relevant synthetic peptide.
Monensin exhibited a profound inhibitory effect on presentation of
308-319 from both viable streptococci and soluble rM5, whereas only
partial reduction of 17-31 presentation was recorded in the same
experiments (Fig. 1B). This data suggested that endosome
acidification is critical for processing of 308-319, whereas
processing of 17-31 was less dependent on endosomal pH.

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Fig. 1.
Effect of inhibitors of endosomal
acidification on presentation of two M5 protein T-cell epitopes.
Presentation of two M5 protein-specific T-cell epitopes (17-31 and
308-319) from viable streptococci (squares), rM5
(diamonds), or synthetic peptides (circles) to
specific T-cell hybridomas, HX17 (closed symbols) and HY2
(open symbols). Different concentrations of ammonium
chloride (A) or monensin (B) are shown as labels
of the x axis. The experiments were performed on at least
three occasions, and data from a representative experiment is
shown.
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Endosomal Proteolysis by Cysteine Proteinases--
There is
evidence to suggest that cysteine proteinases mediate antigen
processing of some model antigens, such as sperm whale myoglobin (8,
21), pigeon cytochrome c (22), tetanus toxin (23), synthetic
oligopeptides (24), and hen egg lysosyme (11) but not ovalbumin (9,
25). To study the role of this class of proteinases in antigen
processing, we used
(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methyl-butane, a membrane permeable inhibitor that reacts with a cysteine residue at
the enzyme active center selectively inactivating cysteine proteinases
(cathepsins L, S, and B) (13, 26). This inhibitor blocked processing of
308-319 from viable streptococci and rM5 and processing of 17-31 from
soluble rM5 but not from bacteria (Fig.
2A). Similarly,
p-hydroxymercuribenzoic acid, an irreversible cysteine
proteinase inhibitor, blocked processing of 308-319 and caused only a
marginal inhibition in presentation of 17-31 from rM5 (Fig.
2B). Thus, processing of viable streptococci for 17-31 presentation was not dependent on cysteine proteinase activity, whereas
this class of intracellular enzymes was critical for processing of
308-319 from bacteria and rM5.

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Fig. 2.
Effect of inhibitors of cysteine proteinases
on presentation of two M5 protein T-cell epitopes. Presentation of
two M5 protein-specific T-cell epitopes (17-31 and 308-319) from
viable streptococci (squares), rM5 (diamonds), or
synthetic peptides (circles) to specific T-cell hybridomas,
HX17 (closed symbols) and HY2 (open symbols).
Different concentrations of
(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methyl-butane (E-64d) (A) or p-hydroxymercuribenzoic
acid (pHMB) (B) are shown as labels of the
x axis. Other details are as in the legend to Fig. 1.
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Involvement of Other Classes of Proteinases in Antigen
Processing--
Data in Fig. 3 show
the effect of other inhibitors on antigen processing.
N
-p-tosyl-L-lysine chloromethyl
ketone (TLCK) and N-tosyl-L-phenylalanine
chloromethyl ketone (TPCK) were used to inactivate trypsin-like and
chymotrypsin-like serine proteinases, respectively (8), and
phenylmethylsulfonyl fluoride to block most serine proteinases. A
peptide aldehyde leupeptin, which is known to react with
serine/cysteine residues at enzyme active centers forming hemiacetal or
hemithioacetal groups, was employed to inactivate both serine and
cysteine proteinases (13) (Fig. 3, A-D). We observed that
TLCK and TPCK exerted a consistent blocking effect on processing of
both epitopes (Fig. 3, A and C).
Phenylmethylsulfonyl fluoride inhibited 308-319 and marginally
suppressed 17-31 presentation (Fig. 3B). The effect of
leupeptin on antigen processing was largely similar to that of
(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methyl-butane in that leupeptin blocked processing of 17-31 from soluble rM5 and
308-319 from both viable bacteria and rM5 (Fig. 3D).
Further data show that 1,10-phenanthroline, which blocks
metalloproteinases, did not interfere with antigen processing of both
epitopes (Fig. 4B). No
cleavage sites recognized by aspartic proteinases were present near
both epitopes (Fig. 5). However,
pepstatin, an acylated pentapeptide isolated from actinomycetes which
is commonly used to irreversibly block aspartic proteinases, inhibited
presentation of 308-319 but not 17-31 (Fig. 4A).

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Fig. 3.
Effect of inhibitors of serine proteinases on
presentation of two M5 protein T-cell epitopes. Presentation of
two M5 protein-specific T-cell epitopes (17-31 and 308-319) from
viable streptococci (squares), rM5 (diamonds), or
synthetic peptides (circles) to specific T-cell hybridomas,
HX17 (closed symbols) and HY2 (open symbols).
Different concentrations of TLCK (A), phenylmethylsulfonyl
fluoride (PMSF) (B), TPCK (C), and
leupeptin (D) are shown as labels of the x axis.
Other details are as in the legend to Fig. 1.
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Fig. 4.
Effect of inhibitors of other intracellular
proteinases on presentation of two M5 protein T-cell epitopes.
Presentation of two M5 protein-specific T-cell epitopes (17-31 and
308-319) from viable streptococci (squares), rM5
(diamonds), or synthetic peptides (circles) to
specific T-cell hybridomas, HX17 (closed symbols) and HY2
(open symbols). Different concentrations of pepstatin (A) or 1,10-phenanthroline (B) are shown as
labels of the x axis. Other details are as in the legend to
Fig. 1.
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Fig. 5.
N-terminal and C-terminal regions of the M5
protein of S. pyogenes containing two immunodominant T-cell
epitopes studied. The underlined sequences correspond
to the synthetic peptides used, and sequences marked in bold
bound the epitope structure. Arrows point to enzyme cleavage
sites as follows: chymtr., chymotrypsin-like serine
proteinases that cleave between F-X, W-X,
Y-X (13, 27); tr., trypsin-like serine
proteinases that cleave between K-X, R-X (13,
27); cath.B, cathepsin B, a cysteine proteinase that cleaves
between K-K, K-R, R-R, or after F-R (13, 30); and cath.S, cathepsin S, a cysteine proteinase that cleaves
between K-L, K-V (31). No aspartic proteinase cleavage sites (cleave between F-F, F-Y, and L-F (13)) were found in the vicinity of either
epitope.
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DISCUSSION |
We have previously shown that like globular proteins, the surface
fibrillar M5 protein of group A streptococci needs intracellular antigen processing for efficient presentation of two immunodominant CD4+ T-cell epitopes (17-31/Ed and
308-319/Ad) to specific T-cell hybridomas (17). Here we
studied the requirements for endosomal proteolytic processing of these
T-cell epitopes by murine macrophages. To this end, we used metabolic
inhibitors of all four classes of intracellular endopeptidases
(cysteine, serine, aspartic, and metalloproteinases). Several
inhibitors with similar specificity at a nontoxic narrow concentration
range were applied to ensure reproducibility of the results. Since the N-terminal epitope (17-31) was bounded by serine proteinase cleavage sites, and both serine and cysteine proteinase sites were located in
the vicinity of the C-terminal epitope (308-319) (Fig. 5), we
hypothesized involvement of predominantly these classes of intracellular enzymes in antigen processing of streptococcal M5 protein.
Evidence was obtained that engagement of a particular class of
proteolytic enzymes in antigen processing of two epitopes on the
streptococcal M5 protein was dependent on both the amino acids flanking
the epitope and the form of antigen delivery (viable bacteria or
soluble rM5). Indeed, our data suggested that processing of 17-31 from
bacteria was mediated by serine proteinases and was not dependent on
endosome acidification, consistent with the presence of serine
proteinase cleavage sites located adjacent to this epitope (Fig. 5) and
with the neutral pH optimum for serine proteinase activity (13, 27).
Interestingly, soluble rM5 protein required both serine and cysteine
proteinases to facilitate processing of 17-31 for presentation to
specific T-cells. In the absence of cysteine proteinase cleavage sites
in the vicinity of 17-31, the effect of cysteine proteinases could be
restricted to Ii chain proteolysis, which is essential for antigen
presentation with newly synthesized MHC-II molecules of the classical
antigen processing pathway (1, 2). Collectively, the data imply that
antigen processing of viable streptococci and soluble rM5 protein for 17-31 presentation occurred largely in different endosomal
compartments, early and late endosomes, respectively. Targeting of rM5
to distinct MHC-II processing compartments in this case would be
expected if the polypeptide chain of this recombinant protein needed
unfolding prior to proteolytic cleavage as has been described for
native but not denatured sperm whale myoglobin (21). In contrast, the N-terminal 17-31 epitope of the M5 protein expressed on the bacterial cell surface could have been cleaved by plasma membrane-associated endopeptidases as described for processing of bovine serum albumin by
the A20 lymphoblastoid cell line (28) and routed to early endosomes. It
is not clear if presentation of 17-31 after processing of whole
bacteria occurred in the context of a large antigen fragment or whether
it was trimmed by serine proteinases after binding to MHC-II molecules
thus protecting the trypsin-like serine proteinase cleavage site within
the epitope (Fig. 5).
Processing of 308-319 from both rM5 and bacteria was mediated by
serine, cysteine, and aspartic proteinases and was found to be
dependent on endosome acidification. The data suggests engagement of
late endosomes/lysosomes that provide the necessary acidic environment
for acquisition of full enzymatic activity of cysteine and aspartic
proteinases (7, 19). Dependence of 308-319 processing on aspartic
proteinases is not consistent with the absence of aspartic proteinase
cleavage sites in the vicinity of this epitope, again suggesting that
aspartic proteinases exerted an indirect effect on antigen processing
via the previously described inhibition of sequential proteolysis of Ii
chain from (
3)Ii3 complexes (2, 4,
29).
Data presented herein indicate that two immunodominant T-cell epitopes
on the streptococcal M5 protein engage different endosomal compartments
and required different classes of intracellular proteolytic enzymes for
antigen processing. Knowledge of the mechanisms of enzymatic cleavage
of protective antigens during antigen processing of T-cell epitopes
from viable bacteria has important implications for live vaccine
delivery systems.
We thank P. Marrack, Howard Hughes Medical
Institute at National Jewish Center, Denver, for BW5147
(TCR


) cells, M. A. Kehoe,
Department of Microbiology for the Manfredo strain of S. pyogenes and recombinant M5 protein, D. Buttle and I. Holen,
Department of Human Metabolism and Clinical Biochemistry, Sheffield
University Medical School, for useful advice on inhibitors, D. Woodland, Department of Immunology, St. Judes Children's Research Hospital, Memphis, for advice on generating hybridomas.