The Giardia intestinalis filamentous cyst wall contains a novel  (1-3)-N-acetyl-D-galactosamine polymer: a structural and conformational study

Gerwig, Gerrit J.; Kuik, J. Albert van; Leeflang, Bas R.; Kamerling, Johannis P.; Vliegenthart, Johannes F.G.; Karr, Craig; Jarroll, Edward L.

doi:10.1093/glycob/cwf059

Cited by 101 publications

(80 citation statements)

References 20 publications

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“…A crucial element in the Giardia life cycle for its survival outside of its host is the cyst wall formation (encystment) that occurs prior to the pathogen exiting the host. The cell wall formed during encystment contains polymers of GalNAc (Gerwig et al, 2002). The enzymes involved in synthesis of amino sugars leading to GalNAc have higher activity in vitro when isolated from encysting Giardia relative to quiescent Giardia (Macechko et al, 1992) and the transcripts for the first five of these enzymes are upregulated during encystment (Lopez et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

“…The outer filamentous wall is made up of proteins (37 %) and carbohydrate filaments (63 %) composed mainly of a [DGalNAc-b(1-3)-D-GalNAc] n homopolymer (Gerwig et al, 2002). The precursor for the GalNAc homopolymer is UDP-GalNAc, which is synthesized from glucose by a pathway of inducible enzymes and amino sugar phosphate intermediates shown in Fig.…”

Section: Introductionmentioning

confidence: 99%

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Amino sugar phosphate levels in Giardia change during cyst wall formation

et al. 2004

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The parasite Giardia intestinalis exists as a trophozoite (vegetative) that infects the human small intestine, and a cyst (infective) that is shed in host faeces. Cyst viability in the environment depends upon a protective cyst wall, which consists of proteins and a unique b(1-3) GalNAc homopolymer. UDP-GalNAc, the precursor for this polysaccharide, is synthesized from glucose by an enzyme pathway that involves amino sugar phosphate intermediates. Using a novel method of microanalysis by capillary electrophoresis, the levels of amino sugar phosphate intermediates in trophozoites before encystment, during a period of active encystment and after the peak of encystment were measured. These levels were used to deduce metabolic control of amino sugar phosphates associated with encystment. Levels of amino sugar phosphate intermediates increased during encystment, and then decreased to nearly non-encysting levels. The most pronounced increase was in glucosamine 6-phosphate, which is the first substrate unique in this pathway, and which is the positive effector for the pathway's putative rate-controlling enzyme, UDP-GlcNAc pyrophosphorylase. Moreover, more UDP-GalNAc than UDP-GlcNAc, its direct precursor, was detected at 24 h. It is postulated that the enhanced UDP-GalNAc is a result of enhanced synthesis of UDP-GlcNAc by the pyrophosphorylase, and its preferential conversion to UDP-GalNAc. These results suggest that kinetics of amino sugar phosphate synthesis in encysting Giardia favours the direction that supports cyst wall synthesis. The enzymes involved in synthesis of UDP-GalNAc and its conversion to cyst wall might be potential targets for therapeutic inhibitors of Giardia infection.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Amino sugar phosphate levels in Giardia change during cyst wall formation

et al. 2004

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“…Because of the extreme pI values of some proteins and presumably also the high sugar (␤1-3 GalNAc homopolymers) content (25) in the preparation, the quality of the separation was comparatively low. For example, none of the CWPs could be resolved, even though they were readily detected in conventional SDS-PAGE and Western analysis (Fig.…”

Section: Enrichment Of Esv-derived Microsomes By Subcellular Fractionmentioning

confidence: 99%

Organelle Proteomics Reveals Cargo Maturation Mechanisms Associated with Golgi-like Encystation Vesicles in the Early-diverged Protozoan Giardia lamblia

Štefanić

Palm

Svärd

et al. 2006

Journal of Biological Chemistry

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During encystationGiardia trophozoites secrete a fibrillar extracellular matrix of glycans and cyst wall proteins on the cell surface. The cyst wall material is accumulated in encystation-specific vesicles (ESVs), specialized Golgi-like compartments generated de novo, after export from the endoplasmic reticulum (ER) and before secretion. These large post-ER vesicles neither have the morphological characteristics of Golgi cisternae nor sorting functions, but may represent an evolutionary early form of the Golgi-like maturation compartment. Because little is known about the genesis and maturation of ESVs, we used a limited proteomics approach to discover novel proteins that are specific for developing ESVs or associated peripherally with these organelles. Unexpectedly, we identified cytoplasmic and luminal factors of the ER quality control system on two-dimensional electrophoresis gels, i.e. several proteasome subunits and HSP70-BiP. We show that BiP is exported to ESVs and retrieved via its C-terminal KDEL signal from ESVs. In contrast, cytoplasmic proteasome complexes undergo a developmentally regulated re-localization to ESVs during encystation. This suggests that maturation of bulk exported cyst wall material in the Golgi-like ESVs involves both continuous activity of ER-associated quality control mechanisms and retrograde Golgi to ER transport.

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“…One of the major components of the outer cyst wall has been identified as a [D-GalNAc(␤133)-D-GalNAc] n homopolymer by chemical methods, mass spectrometry, and hydrogen nuclear magnetic resonance spectroscopy (10). This GalNAc in G. intestinalis is synthesized from endogenous Glc rather than salvaged from external source (5).…”

mentioning

confidence: 99%

Kinetic and Physical Characterization of the Inducible UDP-N-acetylglucosamine Pyrophosphorylase from Giardia intestinalis

Mok

Edwards

2005

Journal of Biological Chemistry

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The UDP-N-acetylglucosamine pyrophosphorylase in Giardia intestinalis (GiUAP) is one of the five inducible enzymes to synthesize UDP-GalNAc, which is an important precursor for cyst wall synthesis. The recombinant UDP-N-acetylglucosamine pyrophosphorylase (rGiUAP) and its mutants G108A and G210A were expressed and identified by SDS-PAGE, size-exclusion chromatography, Western hybridization, and MALDI mass spectrometry. Sequence comparison with other eukaryotic UAPs has identified three specific motifs. Within these motifs alanine substitution for Gly 108 or Gly 210 dramatically reduced the pyrophosphate synthesis, suggesting these amino acids are catalytic residues. Besides, the rGi-UAP was found to have relaxed binding to other uridine-based nucleotides, suggesting the substrate binding pocket is specific to uridine rather than phosphate group(s). Moreover, thermal denaturation analysis showed a significant increase in T m for the rGiUAP and G108A upon binding of the substrate Mg-UTP. In contrast, G210A showed a decreased T m upon binding of Mg-UTP. These results showed that binding of Mg-UTP increases protein stability of the rGiUAP, and the catalytic residue Gly 210 plays a significant role in stabilizing the protein structure. Such stabilization effect induced by substrate binding might be physiologically important as it favors the production of UDP-GlcNAc and hence the downstream GalNAc, which is crucial to survival of Giardia. These results help to define the essential amino acids for catalysis in the GiUAP and reveal the role of Mg-UTP binding in regulation of protein stability.Giardia intestinalis has long been recognized as one of the most early branching eukaryotes (1). It is also one of the most common causes of gastrointestinal infection in human and other mammals (2). When this parasite travels down the intestine in the host, the transformation from a vegetative trophozoite to an infectious cyst requires the synthesis of a rigid cyst wall to survive the adverse environmental conditions outside the host. As this transformation represents a basic adaptive response of a eukaryote to the environment for propagation, G. intestinalis provides a simple eukaryotic model for differentiation and, the biochemical mechanisms of encystation and the cyst wall synthesis have been the subject of intensive investigations in the past decade.Previous studies have shown that cyst wall contains both carbohydrate and protein components (3-9). One of the major components of the outer cyst wall has been identified as a [D-GalNAc(␤133)-D-GalNAc] n homopolymer by chemical methods, mass spectrometry, and hydrogen nuclear magnetic resonance spectroscopy (10). This GalNAc in G. intestinalis is synthesized from endogenous Glc rather than salvaged from external source (5). The precursor of GalNAc, UDP-GalNAc, is synthesized by five inducible enzymes: glucosamine-6-phosphate isomerase (EC 3.5.99.6); glucosamine-6-phosphate N-acetylase (EC 2.3.1.4); phosphoacetylglucosamine mutase (EC 5.4.2.3, AGM) 2 ; UDP-N-acetylglucosamine py...

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The Giardia intestinalis filamentous cyst wall contains a novel (1-3)-N-acetyl-D-galactosamine polymer: a structural and conformational study

Cited by 101 publications

References 20 publications

Amino sugar phosphate levels in Giardia change during cyst wall formation

Amino sugar phosphate levels in Giardia change during cyst wall formation

Organelle Proteomics Reveals Cargo Maturation Mechanisms Associated with Golgi-like Encystation Vesicles in the Early-diverged Protozoan Giardia lamblia

Kinetic and Physical Characterization of the Inducible UDP-N-acetylglucosamine Pyrophosphorylase from Giardia intestinalis

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