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1.  Solution Structure of a Repeated Unit of the ABA-1 Nematode Polyprotein Allergen of Ascaris Reveals a Novel Fold and Two Discrete Lipid-Binding Sites 
Background
Nematode polyprotein allergens (NPAs) are an unusual class of lipid-binding proteins found only in nematodes. They are synthesized as large, tandemly repetitive polyproteins that are post-translationally cleaved into multiple copies of small lipid binding proteins with virtually identical fatty acid and retinol (Vitamin A)-binding characteristics. They are probably central to transport and distribution of small hydrophobic compounds between the tissues of nematodes, and may play key roles in nutrient scavenging, immunomodulation, and IgE antibody-based responses in infection. In some species the repeating units are diverse in amino acid sequence, but, in ascarid and filarial nematodes, many of the units are identical or near-identical. ABA-1A is the most common repeating unit of the NPA of Ascaris suum, and is closely similar to that of Ascaris lumbricoides, the large intestinal roundworm of humans. Immune responses to NPAs have been associated with naturally-acquired resistance to infection in humans, and the immune repertoire to them is under strict genetic control.
Methodology/Principal Findings
The solution structure of ABA-1A was determined by protein nuclear magnetic resonance spectroscopy. The protein adopts a novel seven-helical fold comprising a long central helix that participates in two hollow four-helical bundles on either side. Discrete hydrophobic ligand-binding pockets are found in the N-terminal and C-terminal bundles, and the amino acid sidechains affected by ligand (fatty acid) binding were identified. Recombinant ABA-1A contains tightly-bound ligand(s) of bacterial culture origin in one of its binding sites.
Conclusions/Significance
This is the first mature, post-translationally processed, unit of a naturally-occurring tandemly-repetitive polyprotein to be structurally characterized from any source, and it belongs to a new structural class. NPAs have no counterparts in vertebrates, so represent potential targets for drug or immunological intervention. The nature of the (as yet) unidentified bacterial ligand(s) may be pertinent to this, as will our characterization of the unusual binding sites.
Author Summary
Parasitic nematode worms cause serious health problems in humans and other animals. They can induce allergic-type immune responses, which can be harmful but may at the same time protect against the infections. Allergens are proteins that trigger allergic reactions and these parasites produce a type that is confined to nematodes, the nematode polyprotein allergens (NPAs). These are synthesized as large precursor proteins comprising repeating units of similar amino acid sequence that are subsequently cleaved into multiple copies of the allergen protein. NPAs bind small lipids such as fatty acids and retinol (Vitamin A) and probably transport these sensitive and insoluble compounds between the tissues of the worms. Nematodes cannot synthesize these lipids, so NPAs may also be crucial for extracting nutrients from their hosts. They may also be involved in altering immune responses by controlling the lipids by which the immune and inflammatory cells communicate. We describe the molecular structure of one unit of an NPA, the well-known ABA-1 allergen of Ascaris, and find its structure to be of a type not previously found for lipid-binding proteins, and we describe the unusual sites where lipids bind within this structure.
doi:10.1371/journal.pntd.0001040
PMCID: PMC3079579  PMID: 21526216
2.  Identification of soluble protein fragments by gene fragmentation and genetic selection 
Nucleic Acids Research  2008;36(9):e51.
We describe a new method, which identifies protein fragments for soluble expression in Escherichia coli from a randomly fragmented gene library. Inhibition of E. coli dihydrofolate reductase (DHFR) by trimethoprim (TMP) prevents growth, but this can be relieved by murine DHFR (mDHFR). Bacterial strains expressing mDHFR fusions with the soluble proteins green fluroscent protein (GFP) or EphB2 (SAM domain) displayed markedly increased growth rates with TMP compared to strains expressing insoluble EphB2 (TK domain) or ketosteroid isomerase (KSI). Therefore, mDHFR is affected by the solubility of fusion partners and can act as a reporter of soluble protein expression. Random fragment libraries of the transcription factor Fli1 were generated by deoxyuridine incorporation and endonuclease V cleavage. The fragments were cloned upstream of mDHFR and TMP resistant clones expressing soluble protein were identified. These were found to cluster around the DNA binding ETS domain. A selected Fli1 fragment was expressed independently of mDHFR and was judged to be correctly folded by various biophysical methods including NMR. Soluble fragments of the cell-surface receptor Pecam1 were also identified. This genetic selection method was shown to generate expression clones useful for both structural studies and antibody generation and does not require a priori knowledge of domain architecture.
doi:10.1093/nar/gkn151
PMCID: PMC2396403  PMID: 18420658
3.  THE STRUCTURE OF LEISHMANIA MEXICANA ICP PROVIDES EVIDENCE FOR CONVERGENT EVOLUTION OF CYSTEINE PEPTIDASE INHIBITORS* 
The Journal of biological chemistry  2005;281(9):5821-5828.
Clan CA, family C1 cysteine peptidases (CPs) are important virulence factors and drug targets in parasites that cause neglected diseases. Natural CP inhibitors of the I42 family, known as ICP, occur in some protozoa and bacterial pathogens, but are absent from metazoa. They are active against both parasite and mammalian CPs, despite having no sequence similarity with other classes of CP inhibitor. Recent data suggest that L. mexicana ICP plays an important role in host-parasite interactions. We have now solved the structure of ICP from L. mexicanaby NMR and shown that it adopts a type of immunoglobulin-like fold not previously reported in lower eukaryotes or bacteria. The structure places three loops containing highly conserved residues at one end of the molecule, one loop being highly mobile. Interaction studies with CPs confirm the importance of these loops for the interaction between ICP and CPs and suggest the mechanism of inhibition. Structure-guided mutagenesis of ICP has revealed that residues in the mobile loop are critical for CP inhibition. Data-driven docking models support the importance of the loops in the ICP-CP interaction. This study provides structural evidence for the convergent evolution from an immunoglobulin fold of CP inhibitors with a cystatin-like mechanism.
doi:10.1074/jbc.M510868200
PMCID: PMC1473161  PMID: 16407198

Results 1-3 (3)