Cyst Wall Protein 1 Is a Lectin That Binds to Curled Fibrils of the GalNAc Homopolymer


The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique β-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWPLRR) and a C-terminal conserved Cys-rich region (CWPCRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (∼400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (∼1.2 µm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1LRR. In contrast, neither MBP alone nor MBP fused to CWP1CRR bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase.


Vyšlo v časopise: Cyst Wall Protein 1 Is a Lectin That Binds to Curled Fibrils of the GalNAc Homopolymer. PLoS Pathog 6(8): e32767. doi:10.1371/journal.ppat.1001059
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1001059

Souhrn

The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique β-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWPLRR) and a C-terminal conserved Cys-rich region (CWPCRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (∼400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (∼1.2 µm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1LRR. In contrast, neither MBP alone nor MBP fused to CWP1CRR bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase.


Zdroje

1. AdamRD

2001 Biology of Giardia lamblia. Clin Microbiol Rev 14 447 475

2. MorrisonHG

McArthurAG

GillinFD

AleySB

AdamRD

2007 Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science 317 1921 1926

3. FranzénO

Jerlström-HultqvistJ

CastroE

SherwoodE

AnkarklevJ

2009 Draft genome sequencing of Giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different species? PLoS Pathog 5 e1000560

4. SteinerTS

ThielmanNM

GuerrantRL

1997 Protozoal agents: what are the dangers for the public water supply? Annu Rev Med 48 329 340

5. LauwaetT

DavidsBJ

ReinerDS

GillinFD

2007 Encystation of Giardia lamblia: a model for other parasites. Curr Opin Microbiol 210 554 559

6. LujanHD

MowattMR

ConradJT

BowersB

NashTE

1995 Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats. Implications for secretory granule formation and protein assembly into the cyst wall. J Biol Chem 270 29307 29313

7. MowattMR

LujanHD

CottenDB

BowersB

YeeJ

1995 Developmentally regulated expression of a Giardia lamblia cyst wall protein gene. Mol Microbiol 15 955 963

8. HehlAB

MartiM

KohlerP

2000 Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia. Mol Biol Cell 11 1789 1800

9. SunCH

McCafferyJM

ReinerDS

GillinFD

2003 Mining the Giardia lamblia genome for new cyst wall proteins. J Biol Chem 278 21701 21708

10. MengTC

HetskoML

GillinFD

1996 Inhibition of Giardia lamblia excystation by antibodies against cyst walls and by wheat germ agglutinin. Infect Immun 64 2151 2157

11. BooneJH

WilkinsTD

NashTE

BrandonJE

MaciasEA

1999 TechLab and alexon Giardia enzyme-linked immunosorbent assay kits detect cyst wall protein 1. J Clin Microbiol 37 611 614

12. LeeP

Abdul-WahidA

FaubertGM

2009 Comparison of the local immune response against Giardia lamblia cyst wall protein 2 induced by recombinant Lactococcus lactis and Streptococcus gordonii. Microbes Infect 11 20 28

13. Ortega-BarriaE

WardHD

EvansJE

PereiraME

1990 N-acetyl-D-glucosamine is present in cysts and trophozoites of Giardia lamblia and serves as receptor for wheatgerm agglutinin. Mol Biochem Parasitol 43 151 165

14. SamuelsonJ

BanerjeeS

MagnelliP

CuiJ

KelleherDJ

2005 The diversity of protist and fungal dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases. Proc Natl Acad Sci USA 102 1548 1553

15. RatnerDM

CuiJ

SteffenM

MooreLL

RobbinsPW

2008 Changes in the N-glycome, glycoproteins with Asn linked glycans, of Giardia lamblia with differentiation from trophozoites to cysts. Eukaryot Cell 7 1930 1940

16. DavidsBJ

ReinerDS

BirkelandSR

PreheimSP

CiprianoMJ

2006 A new family of giardial cysteine-rich non-VSP protein genes and a novel cyst protein. PLoS ONE 1 e44

17. ChiuPW

HuangYC

PanYJ

WangCH

SunCH

2010 A novel family of cyst proteins with epidermal growth factor repeats in Giardia lamblia. PLoS Negl Trop Dis 45 e677

18. StefanicS

MorfL

KulangaraC

RegosA

SondaS

2009 Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote. J Cell Sci 122 2846 2856

19. GottigN

EliasEV

QuirogaR

NoresMJ

SolariAJ

2006 Active and passive mechanisms drive secretory granule biogenesis during differentiation of the intestinal parasite Giardia lamblia. J Biol Chem 281 18156 18166

20. KonradC

SpycherC

HehlAB

2010 Selective condensation drives partitioning and sequential secretion of cyst wall proteins in differentiating Giardia lamblia. PLoS Pathog 6 e1000835

21. TouzMC

NoresMJ

SlavinI

CarmonaC

ConradJT

2002 The activity of a developmentally regulated cysteine proteinase is required for cyst wall formation in the primitive eukaryote Giardia lamblia. J Biol Chem 277 8474 8481

22. WardW

AlvaradoL

RawlingsND

EngelJC

FranklinC

1997 A primitive enzyme for a primitive cell: the protease required for excystation of Giardia. Cell 89 437 444

23. BoucherSE

GillinFD

1990 Excystation of in vitro-derived Giardia lamblia cysts. Infect Immun 58 3516 3522

24. ReinerDS

McCafferyJM

GillinFD

2001 Reversible interruption of Giardia lamblia cyst wall protein transport in a novel regulated secretory pathway. Cell Microbiol 3 459 472

25. DavidsBJ

MehtaK

FesusL

McCafferyJM

GillinFD

2004 Dependence of Giardia lamblia encystation on novel transglutaminase activity. Mol Biochem Parasitol 136 173 180

26. SlavinI

SauraA

CarranzaPG

TouzMC

NoresMJ

2002 Dephosphorylation of cyst wall proteins by a secreted lysosomal acid phosphatase is essential for excystation of Giardia lamblia. Mol Biochem Parasitol 122 95 98

27. LauwaetT

DavidsBJ

Torres-EscobarA

BirkelandSR

CiprianoMJ

2007 Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation. Mol Biochem Parasitol 152 80 89

28. GerwigGJ

van KuikJA

LeeflangBR

KamerlingJP

VliegenthartJF

2002 The Giardia intestinalis filamentous cyst wall contains a novel beta(1-3)-N-acetyl-D-galactosamine polymer: a structural and conformational study. Glycobiology 12 499 505

29. ErlandsenSL

MacechkoPT

van KeulenH

JarrollEL

1996 Formation of the Giardia cyst wall: studies on extracellular assembly using immunogold labeling and high resolution field emission SEM. J Eukaryot Microbiol 43 416 429

30. Chávez-MunguíaB

Cedillo-RiveraR

Martínez-PalomoA

2004 The ultrastructure of the cyst wall of Giardia lamblia. J Eukaryot Microbiol 51 220 226

31. KarrCD

JarrollEL

2004 Cyst wall synthase: N-acetylgalactosaminyltransferase activity is induced to form the novel N-acetylgalactosamine polysaccharide in the Giardia cyst wall. Microbiology 150 1237 1243

32. WestCM

2003 Comparative analysis of spore coat formation, structure, and function in Dictyostelium. Int Rev Cytol 222 237 293

33. LesageG

BusseyH

2006 Cell wall assembly in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 70 317 343

34. JametE

AlbenneC

BoudartG

IrshadM

CanutH

2008 Recent advances in plant cell wall proteomics. Proteomics 8 893 908

35. FrisardiM

GhoshSK

FieldJ

Van DellenK

RogersR

2000 The most abundant glycoprotein of amebic cyst walls (Jacob) is a lectin with five Cys-rich, chitin-binding domains. Infect Immun 68 4217 4224

36. Van DellenK

GhoshSK

RobbinsPW

LoftusB

SamuelsonJ

2002 Entamoeba histolytica lectins contain unique 6-Cys or 8-Cys chitin-binding domains. Infect Immun 70 3259 3263

37. de la VegaH

SpechtCA

SeminoCE

RobbinsPW

EichingerD

1997 Cloning and expression of chitinases of Entamoebae. Mol Biochem Parasitol 85 139 147

38. Van DellenKL

ChatterjeeA

RatnerDM

MagnelliPE

CipolloJF

2006 Unique posttranslational modifications of chitin-binding lectins of Entamoeba invadens cyst walls. Eukaryot Cell 5 836 848

39. ChatterjeeA

GhoshSK

JangK

BullittE

MooreL

2009 Evidence for a “wattle and daub” model of the cyst wall of entamoeba. PLoS Pathog 5 e1000498

40. MannersDJ

MeyerMT

1977 The molecular structures of some glucans from the cell walls of Schizosaccharomyces pombe. Carbohydr Res 57 189 203

41. NallamsettyS

WaughDS

2007 A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag. Nat Protoc 2 383 391

42. CabibE

RohDH

SchmidtM

CrottiLB

VarmaA

2001 The yeast cell wall and septum as paradigms of cell growth and morphogenesis. J Biol Chem 276 19679 19682

43. NanduriJ

WilliamsS

AjiT

FlaniganTP

1999 Characterization of an immunogenic glycocalyx on the surfaces of Cryptosporidium parvum oocysts and sporozoites. Infect Immun 67 2022 2024

44. OliverRM

1973 Negative stain electron microscopy of protein macromolecules. Methods Enzymol 27 616 762

45. ChatterjeeA

BanerjeeS

SteffenM

O'ConnorRM

WardHD

2010 Evidence for mucin-like glycoproteins that tether sporozoites of Cryptosporidium parvum to the inner surface of the oocyst wall. Eukaryot Cell 9 84 96

46. GhoshS

FrisardiM

RogersR

SamuelsonJ

2001 How Giardia swim and divide. Infect Immun 69 7866 7872

47. DingSY

HimmelME

2006 The maize primary cell wall microfibril: a new model derived from direct visualization. J Agric Food Chem 54 597 606

48. LenardonMD

WhittonRK

MunroCA

MarshallD

GowNA

2007 Individual chitin synthase enzymes synthesize microfibrils of differing structure at specific locations in the Candida albicans cell wall. Mol Microbiol 66 1164 1173

49. WrightHT

SandrasegaramG

WrightCS

1991 Evolution of a family of N-acetylglucosamine binding proteins containing the disulfide-rich domain of wheat germ agglutinin. J Mol Evol 33 283 294

50. JinMS

LeeJO

2008 Structures of the toll-like receptor family and its ligand complexes. Immunity 29 182 191

51. ShawMH

ReimerT

KimYG

NuñezG

2008 NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Curr Opin Immunol 20 377 382

52. CabibE

ShematuckEM

1981 Structural polysaccharides of plants and fungi: comparative and morphogenetic aspects.

GinsburgV

RobbinsP

Biology of Carbohydrates vol1 New York John Wiley & Sons 51 90

53. CarringtonWA

LynchRM

MooreED

IsenbergG

FogartyKE

1995 Superresolution three-dimensional images of fluorescence in cells with minimal light exposure. Science 268 1483 1487

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2010 Číslo 8
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa