Total and Lectin-Binding Proteome of Spherulin from Coccidioides posadasii.

Coccidioides is a virulent dimorphic fungus that causes coccidioidomycosis (valley fever) in mammals, including humans. Although the genome has been sequenced, a proteomic analysis does not exist. To address this gap in proteomic knowledge, we generated the proteome of spherulin (a well-studied lysate of fungal spherules) and identified 1390 proteins. Some of the proteins included glycosylation enzymes, which led us to hypothesize that fungal glycosylation patterns may be different from those of mammals and could be exploited to detect Coccidioides in tissues. We performed lectin-based immunohistochemistry on formalin-fixed paraffin-embedded human patients' lung tissues. GSL-II (Griffonia simplificonia lectin II) and sWGA (succinylated wheat germ agglutinin) lectins bound specifically to endospores and spherules in infected lungs. To identify lectin-binding glycoproteins in spherulin, we performed lectin-affinity chromatography, followed by LC-MS/MS. A total of 195 glycoproteins from spherulin bound to GSL-II, 224 glycoproteins bound to sWGA, and 145 glycoproteins bound to both lectins. This is the first report of the specific reactivity of GSL-II and sWGA lectins to Coccidioides endospores and spherules in infected human tissues and the first listing of the Coccidioidal proteome from spherulin using sequences present in three Coccidioides databases: RefSeq, SwissProt, and The Broad Institute's Coccidioides Genome project.

Sph3 Is a Glycoside Hydrolase Required for the Biosynthesis of Galactosaminogalactan in Aspergillus fumigatus.

Aspergillus fumigatus is the most virulent species within the Aspergillus genus and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis, we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (ß/α)8 fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, Asp-166, Glu-167, and Glu-222, that are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of Asp-166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GH135.

Microbial relatives of the seed storage proteins of higher plants: conservation of structure and diversification of function during evolution of the cupin superfamily.

This review summarizes the recent discovery of the cupin superfamily (from the Latin term "cupa," a small barrel) of functionally diverse proteins that initially were limited to several higher plant proteins such as seed storage proteins, germin (an oxalate oxidase), germin-like proteins, and auxin-binding protein. Knowledge of the three-dimensional structure of two vicilins, seed proteins with a characteristic beta-barrel core, led to the identification of a small number of conserved residues and thence to the discovery of several microbial proteins which share these key amino acids. In particular, there is a highly conserved pattern of two histidine-containing motifs with a varied intermotif spacing. This cupin signature is found as a central component of many microbial proteins including certain types of phosphomannose isomerase, polyketide synthase, epimerase, and dioxygenase. In addition, the signature has been identified within the N-terminal effector domain in a subgroup of bacterial AraC transcription factors. As well as these single-domain cupins, this survey has identified other classes of two-domain bicupins including bacterial gentisate 1, 2-dioxygenases and 1-hydroxy-2-naphthoate dioxygenases, fungal oxalate decarboxylases, and legume sucrose-binding proteins. Cupin evolution is discussed from the perspective of the structure-function relationships, using data from the genomes of several prokaryotes, especially Bacillus subtilis. Many of these functions involve aspects of sugar metabolism and cell wall synthesis and are concerned with responses to abiotic stress such as heat, desiccation, or starvation. Particular emphasis is also given to the oxalate-degrading enzymes from microbes, their biological significance, and their value in a range of medical and other applications.

Evolutionary relationships of the metazoan beta gamma-crystallins, including that from the marine sponge Geodia cydonium.

beta gamma-crystallins are one major component of vertebrate lenses. Here the isolation and characterization of a cDNA, coding for the first beta gamma-crystallin molecule from an invertebrate species, the marine sponge Geodia cydonium, is described. The size of the transcript as determined by Northern blotting was 0.7 kb in length. The deduced amino acid sequence consists of 163 aa residues and comprises four repeated motifs which compose the two domains of the beta gamma-crystallin. Motif 3 contains the characteristic beta gamma-crystallin 'Greek key' motif signature, while in each of the three other repeats, one aa residue is replaced by an aa with the same physico-chemical property. The sponge peptide shows striking similarities to vertebrate beta gamma-crystallins. Analysis by neighbour joining of the sponge motifs with the two motifs present in spherulin 3a of Physarum polycephalum shows that motif 4 of the sponge beta gamma-crystallin was added as the last single sequence to the tree. The data support the view that the beta gamma-crystallin superfamily, present in eukaryotes, evolved from a common ancestor including also the sponge beta gamma-crystallin.