LBP/BPI proteins and their relatives: conservation over evolution and roles in mutualism

LBP [LPS (lipopolysaccharide)-binding protein] and BPI (bactericidal/permeability-increasing protein) are components of the immune system that have been principally studied in mammals for their involvement in defence against bacterial pathogens. These proteins share a basic architecture and residues involved in LPS binding. Putative orthologues, i.e. proteins encoded by similar genes that diverged from a common ancestor, have been found in a number of non-mammalian vertebrate species and several non-vertebrates. Similar to other aspects of immunity, such as the activity of Toll-like receptors and NOD (nucleotide-binding oligomerization domain) proteins, analysis of the conservation of LBPs and BPIs in the invertebrates promises to provide insight into features essential to the form and function of these molecules. This review considers state-of-the-art knowledge in the diversity of the LBP/BPI proteins across the eukaryotes and also considers their role in mutualistic symbioses. Recent studies of the LBPs and BPIs in an invertebrate model of beneficial associations, the Hawaiian bobtail squid Euprymna scolopes' alliance with the marine luminous bacterium Vibrio fischeri, are discussed as an example of the use of non-vertebrate models for the study of LBPs and BPIs.

Molecular cloning and biochemical characterization of sialidases from zebrafish (Danio rerio)

Sialidases remove sialic acid residues from various sialo-derivatives. To gain further insights into the biological roles of sialidases in vertebrates, we exploited zebrafish (Danio rerio) as an animal model. A zebrafish transcriptome- and genome-wide search using the sequences of the human NEU polypeptides as templates revealed the presence of seven different genes related to human sialidases. neu1 and neu4 are the putative orthologues of the mammalian sialidases NEU1 and NEU4 respectively. Interestingly, the remaining genes are organized in clusters located on chromosome 21 and are all more closely related to mammalian sialidase NEU3. They were thus named neu3.1, neu3.2, neu3.3, neu3.4 and neu3.5. Using RT–PCR (reverse transcription–PCR) we detected transcripts for all genes, apart from neu3.4, and whole-mount in situ hybridization experiments show a localized expression pattern in gut and lens for neu3.1 and neu4 respectively. Transfection experiments in COS7 (monkey kidney) cells demonstrate that Neu3.1, Neu3.2, Neu3.3 and Neu4 zebrafish proteins are sialidase enzymes. Neu3.1, Neu3.3 and Neu4 are membrane-associated and show a very acidic pH optimum below 3.0, whereas Neu3.2 is a soluble sialidase with a pH optimum of 5.6. These results were further confirmed by subcellular localization studies carried out using immunofluorescence. Moreover, expression in COS7 cells of these novel zebrafish sialidases (with the exception of Neu3.2) induces a significant modification of the ganglioside pattern, consistent with the results obtained with membrane-associated mammalian sialidases. Overall, the redundancy of sialidases together with their expression profile and their activity exerted on gangliosides of living cells indicate the biological relevance of this class of enzymes in zebrafish.

Implications of a genomic search for autophagy-related genes in trypanosomatids

Autophagy is the process by which cellular components are directed to and degraded in the vacuole or lysosome and has been studied largely in yeasts. We present here an in silico genomic analysis of trypanosomatid autophagy aimed at highlighting similarities and differences with autophagy in other organisms. Less than half of the yeast autophagy-related proteins examined have certain putative orthologues in trypanosomatids. A cytosol-to-vacuole transport system is clearly lacking in these organisms. Other absences are even more unexpected and have implications for our understanding of the molecular mechanisms of autophagy. The results are consistent with taxon-specific addition of components to a core autophagy machinery during evolution.

Analysis of Telomerase in Candida albicans: Potential Role in Telomere End Protection

ABSTRACT Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of telomere terminal repeats. Analysis of the telomerase complex in the budding yeast Saccharomyces cerevisiae has revealed the presence of one catalytic protein subunit (Est2p/TERT) and at least two noncatalytic components (Est1p and Est3p). The genome of the pathogenic yeast Candida albicans contains putative orthologues of all three telomerase components. Disruption of each homologue resulted in significant but distinct telomere dysfunction in Candida. Similar to S. cerevisiae, the Candida EST3 disruption strain exhibits progressive telomere loss over many generations, at a rate that is consistent with incomplete replication. In contrast, telomeres in both the Candida TERT and EST1 disruption strains can contract rapidly, followed by partial or nearly complete recovery, suggesting a defect in telomere “capping.” We propose that these two telomerase subunits may participate in the protection of chromosomal ends in Candida. Analysis of telomerase-mediated primer extension in vitro indicates that only the TERT protein is absolutely essential for enzyme activity. Our results support the conservation of telomerase protein components beyond the catalytic subunit but reveal species-specific phenotypic alterations in response to loss of individual telomerase component. We also identify potential homologues of Est1p in phylogenetically diverse organisms. The Est1p sequence family possesses a conserved N-terminal domain predicted to be structurally related to tetratricopeptide repeat-containing proteins.