Microbial carbohydrate depolymerization by
antigen-presenting cells: Deamination prior to
presentation by the MHCII pathway
Jinyou Duan, Fikri Y. Avci, and Dennis L. Kasper*
Department of Medicine, Channing Laboratory, Brigham and Women’s Hospital and Department of Microbiology and Molecular Genetics, Harvard Medical
School, Boston, MA 02115
Communicated by John B. Robbins, National Institutes of Health, Bethesda, MD, February 1, 2008 (received for review October 4, 2007)
After uptake by the endosome of an antigen-presenting cell (APC),
exogenous proteins are known to be degraded into peptides by
protease digestion. Here, we report the mechanism by which pure
carbohydrates can be depolymerized within APC endosomes/lyso-
somes by nitric oxide (NO)-derived reactive nitrogen species (RNSs)
and/or superoxide-derived reactive oxygen species (ROSs). Earlier
studies showed that depolymerization of polysaccharide A (PSA)
from Bacteroides fragilis in the endosome depends on the APC’s
having an intact inducible nitric oxide synthase (iNOS) gene; the
chemical mechanism underlying depolymerization of a carbohy-
drate within the endosome/lysosome is described here. Examining
the ability of the major RNSs to degrade PSA, we determined that
deamination is the predominant mechanism for PSA processing in
APCs and is a required step in PSA presentation to CD4?T cells by
MHCII molecules. Structural characterization of the NO-derived
product PSA-NO indicates that partial deaminative depolymeriza-
tion does not alter the zwitterionic nature of PSA. Unlike native
PSA, PSA-NO is presented by iNOS-deficient APCs to induce CD4?
T cell proliferation. Furthermore, metabolically active APCs are
required for PSA-NO presentation. In contrast to PSA degradation
by RNSs, dextran depolymerization in the endosome depends on
ROSs, including hydrogen peroxide- and superoxide-derived ROSs.
This study provides evidence that MHCII pathway-mediated car-
bohydrate antigen processing in APCs is achieved by chemical
reactions. RNSs and ROSs may be involved in the presentation of
glycopeptides by MHC molecules via the processing of other
carbohydrate-containing antigens, such as bacterial or viral glyco-
proteins or glycoconjugate vaccines.
antigen processing ? MHC class II ? reactive nitrogen species
on the surface of many pathogenic bacteria are carbohydrates
traditionally thought to stimulate an immune response that, in all
cases, was assumed to be T cell independent (3). However, our
earlier studies showed that polysaccharides with a zwitterionic
charge motif can indeed activate CD4?T cells after processing
and presentation through the MHCII pathway (1). Information
has been lacking on the chemical mechanism(s) underlying this
In terms of T cell activation, polysaccharide A (PSA) [its
structure as shown in supporting information (SI) Fig. S1], the
and the best characterized ZPS (4, 5), has profound biological
importance. During intestinal colonization in mice, PSA-
expressing B. fragilis directs maturation of the mammalian
immune system. Mono-association of germ-free mice with a WT
PSA-bearing B. fragilis strain—but not with an isogenic mutant
incapable of synthesizing PSA—corrects systemic T cell defi-
ciencies, redresses Th1/Th2 imbalances, and directs lymphoid
organogenesis (5). Toll-like receptor 2 (TLR2) coordinates an
innate and adaptive immune response to PSA (a TLR2 agonist)
that results in production of IFN ?—a key factor in the Th1
differentiation observed in colonization studies (6).
Once within the APC endosomes/lysosomes, PSA is degraded
to a smaller molecule (?10–15 kDa) before being presented to
CD4?T cells (1). The surprising dependence of this degradation
on inducible nitric oxide synthase (iNOS) suggests a processing
mechanism distinct from the enzymatic cleavage responsible for
cellular processing of protein antigens. Given the established
importance of PSA and the lack of information on processing of
this ZPS or any other carbohydrate, we sought to define the
chemical mechanism(s) responsible for PSA processing. An
in-depth understanding of this key process may elucidate non-
enzymatic processing of numerous antigens through this
Degradation of Carbohydrates Within APC Endosomes by Reactive
Nitrogen Species (RNSs) and Reactive Oxygen Species (ROSs). After
exposure of APCs to cytokines or microbial products, iNOS is
up-regulated and generates large quantities of nitric oxide (NO)
by catalyzing the oxidation of L-arginine (7). NO is a short-lived
radical that forms various NO-derived RNSs. To delineate the
mechanism of NO-dependent processing of PSA in APCs, we
identified the role of iNOS in the processing of PSA in CD11c?
dendritic cells (DCs). It had been shown that iNOS is required
for PSA processing by total splenic mononuclear cells, but no
work had been performed on processing specifically in DCs, the
most relevant APCs for presenting PSA to T cells.
After 72 h of uptake and processing, PSA is degraded to a
different extent in WT and in iNOS?/?DCs (Fig. 1A), and
N-acetyl PSA (the fully N-acetylated product of PSA) is de-
in response to external stimuli, ROSs and RNSs produce syn-
ergistic and/or antagonistic effects in APCs (8), these data
suggest that, in addition to RNSs, other reactive species (for
instance, ROSs) in APCs may effectively process polysaccha-
rides with a zwitterionic charge (PSA) or a negative charge
(N-acetyl PSA). Furthermore, the substantially greater suppres-
sion of PSA degradation in iNOS?/?DCs than in WT DCs (Fig.
1A) indicates that RNSs are the predominant reactive species
responsible for PSA processing. However, N-acetyl PSA pro-
cessing in WT DCs does not differ significantly from that in
iNOS?/?DCs (Fig. 1B). Because deaminative depolymerization
at free amino or N-sulfo groups—but not at N-acetyl groups—of
Author contributions: J.D., F.Y.A., and D.L.K. designed research; J.D. and F.Y.A. performed
research; J.D., F.Y.A., and D.L.K. contributed new reagents/analytic tools; J.D., F.Y.A., and
D.L.K. analyzed data; and J.D., F.Y.A., and D.L.K. wrote the paper.
The authors declare no conflict of interest.
*To whom correspondence should be addressed. E-mail: email@example.com.
© 2008 by The National Academy of Sciences of the USA
April 1, 2008 ?
vol. 105 ?
no. 13 ?
agarose gel and visualized with ethidium bromide.
NO Detection. WT or iNOS?/?splenic DCs (?3 ? 106/ml) with PSA (500 ?g/ml)
or LPS (1 ?g/ml) were stimulated for 72 h. Nitrite production in the cell
supernatants was measured with the Griess Reagent System.
In Vitro T Cell Proliferation Assays. Human T cell proliferation assays were
and 1 ? 105irradiated MNCs (?3,300 rad) were cocultured in 200 ?l of
modified RPMI medium 1640 (American Type Culture Collection) supple-
mented with 10% FBS and 1% penicillin–streptomycin. SEA (10 ng/ml) was
used as a positive control. On day 6, cells were pulsed with [3H]thymidine and
harvested 8 h later. Radioactive uptake was measured by liquid scintillation.
In blocking experiments with human cells, colchicine (1 ?M), brefeldin A (100
?g/ml), or mAbs (1.5 ?g/ml) to HLA-A, -B, and -C; HLA-DR, -DQ, and -DP; and
isotype controls (IgG2a and IgG3) were cocultured with cells 30 min before
addition of PSA-NO (100 ?g/ml).
For the mouse T cell proliferation assay, PSA (100 ?g/ml), PSA-NO (20
?g/ml), and SEA (10 ng/ml) were added to the coculture of 1 ? 105irradiated
DCs and 2 ? 105CD4?T cells (200 ?l per well). For blocking experiments with
mouse cells, colchicine (0.5 ?M), brefeldin A (50 ?g/ml), or mAb (1.5 ?g/ml) to
of PSA-NO (20 ?g/ml). In fixation experiments, DCs were fixed with 2%
formaldehyde in PBS at 4°C for 30 min, and fixed DCs were washed twice with
cold PBS before coculture with CD4?T cells. After 4 days, T cell proliferation
was assayed as above.
Mouse Model for Intraabdominal Abscess Formation. In an intraabdominal
abscess model (1, 29), mice were injected i.p. with 1? PBS (control), PSA (50
?g), or PSA-NO (50 ?g) mixed with sterile cecal contents (SCC). Animals were
Statistical Analyses. Abscess induction differences between groups were eval-
uated by Fisher’s exact test (Instat; GraphPad Software). Means from T cell
proliferation assays were compared by unpaired t test.
ACKNOWLEDGMENTS. We thank Ms. Jaylyn Olivo and Ms. Julie McCoy for
helpful editorial assistance and Drs. Sarkis K. Mazmanian and Rachel
RO1 AI039576 (to D.L.K.).
1. Cobb BA, Wang Q, Tzianabos AO, Kasper DL (2004) Polysaccharide processing and
presentation by the MHCII pathway. Cell 117:677–687.
responses. Cell 126:847–850.
3. Chan WK, Law HK, Lin ZB, Lau YL, Chan GC (2007) Response of human dendritic cells
to different immunomodulatory polysaccharides derived from mushroom and barley.
Int Immunol 19:891–899.
4. Baumann H, Tzianabos AO, Brisson JR, Kasper DL, Jennings HJ (1992) Structural
elucidation of two capsular polysaccharides from one strain of Bacteroides fragilis
using high-resolution NMR spectroscopy. Biochemistry 31:4081–4089.
5. Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL (2005) An immunomodulatory
molecule of symbiotic bacteria directs maturation of the host immune system. Cell
6. Wang Q, et al. (2006) A bacterial carbohydrate links innate and adaptive responses
through Toll-like receptor 2. J Exp Med 203:2853–2863.
7. Kolios G, Valatas V, Ward SG (2004) Nitric oxide in inflammatory bowel disease: A
universal messenger in an unsolved puzzle. Immunology 113:427–437.
8. Bru ¨neB(2005)Theintimaterelationbetweennitricoxideandsuperoxideinapoptosis
and cell survival. Antioxid Redox Signal 7:497–507.
9. Hassan MS, Mileva MM, Dweck HS, Rosenfeld L (1998) Nitric Oxide products degrade
chrondroitin sulfate. Nitric Oxide 2:360–365.
11. Kumar AP, Ryan C, Cordy V, Reynolds WF (2005) Inducible nitric oxide synthase
expression is inhibited by myeloperoxidase. Nitric Oxide 13:42–53.
12. Li M, Rosenfeld L, Vilar RE, Cowman MK (1997) Degradation of hyaluronan by per-
oxynitrite. Arch Biochem Biophys 341:245–250.
13. Wong SF, Halliwell B, Richmond R, Skowreneck WR (1981) The role of superoxide and
hydroxyl radicals in the degradation of hyaluronic acid induced by metal ions and by
ascorbic acid. J Inorg Biochem 14:127–134.
14. Wink DA, et al. (1998) The cytotoxicity of nitroxyl: Possible implications for the
pathophysiological role of NO. Arch Biochem Biophys 351:66–74.
15. Miranda KM, et al. (2005) Mechanism of aerobic decomposition of Angeli’s salt
(sodium trioxodinitrate) at physiological pH. J Am Chem Soc 127:722–731.
16. Vig M, et al. (2004) Inducible nitric oxide synthase in T cells regulates T cell death and
immune memory. J Clin Invest 113:1734–1742.
induced by pathogenic bacteria. J Bio Chem 275:6733–6740.
18. Prigozy TI, et al. (2001) Glycolipid antigen processing for presentation by CD1d
molecules. Science 291:664–667.
19. Mani K, Cheng F, Fransson LA (2007) Heparan sulfate degradation products can
associate with oxidized proteins and proteasomes. J Biol Chem 282:21934–21944.
20. Kaufmann SH, Schaible UE (2005) Antigen presentation and recognition in bacterial
infections. Curr Opin Immunol 17:79–87.
21. Wong SH, Santambrogio L, Strominger JL (2004) Caspases and nitric oxide broadly
Natl Acad Sci USA 101(51):17783–17788.
22. Vlad AM, et al. (2002) Complex carbohydrates are not removed during Processing of
glycoproteins by dendritic cells: Processing of tumor antigen MUC1 glycopeptides for
presentation to major histocompatibility complex class II-restricted T cells. J Exp Med
23. Ceppellini R, et al. (1989) Binding of labelled influenza matrix peptide to HLA DR in
living B lymphoid cells. Nature 339:392–394.
24. Shively JE, Conrad HE (1976) Formation of anhydrosugars in the chemical depolymer-
ization of heparin. Biochemistry 15:3932–3942.
25. Kenne L, Lindberg B, Unger P, Gustafsson B, Holme T (1982) Structural studies of the
Vibrio cholerae O-antigen. Carbohydr Res 100:341–349.
polysaccharide from Streptococcus pneumoniae type 1. Carbohydr Res 78:111–117.
II-dependent interactions. J Immunol 169:6149–6153.
28. Gomez-Ambrosi J, et al. (2004) Reduced adipose tissue mass and hypoleptinemia in
iNOS deficient mice: Effect of LPS on plasma leptin and adiponectin concentrations.
FEBS Lett 577:351–356.
29. Chung DR, et al. (2002) CD4? T cells regulate surgical and postinfectious adhesion
formation. J Exp Med 195:1471–1478.
www.pnas.org?cgi?doi?10.1073?pnas.0800974105Duan et al.