Hpa2 Gene Cloning.

From 1999-2003, Oxford GlycoSciences (OGS) ran a successful drug discovery oncology programme to discover small molecule inhibitors of the Heparanase I enzyme (HPSE1). HPSE1 at the time was widely regarded as being the sole mammalian enzyme capable of cleaving Heparan Sulfate (HS). A second family protein member however called Heparanase 2 (HPSE2) including splice forms was subsequently discovered by PCR analysis based on EST sequences. HPSE2 was found to be expressed mainly in smooth muscle containing tissues, particularly bladder and brain. HPSE2 is poorly expressed in haematopoietic cells and placenta which contrasts with the HPSE1 distribution pattern. HPSE2 binds more strongly to HS than HPSE1 and is believed to out compete for substrate binding and so in effect act as a tumor suppressor. So far, all attempts to show specific HPSE2 endoglycosidase activity against HS have failed suggesting that the enzyme may act as a pseudoenzyme that has evolved to retain only certain non-catalytic heparanase like functions. A breakthrough in the elucidation of functional roles for HPSE2 came about in 2010 with the linkage of HPSE2 gene deletions and mutations to the development of Ochoa/Urofacial Syndrome. Future work into the mechanistic analysis of HPSE2's role in signalling, tumor suppression and bladder/nerve functioning are needed to fully explore the role of this family of proteins.

An Atlas of ß-Glucuronidases in the Human Intestinal Microbiome.

Microbiome-encoded ß-glucuronidase (GUS) enzymes play important roles in human health by metabolizing drugs in the gastrointestinal (GI) tract. The numbers, types, and diversity of these proteins in the human GI microbiome, however, remain undefined. We present an atlas of GUS enzymes comprehensive for the Human Microbiome Project GI database. We identify 3,013 total and 279 unique microbiome-encoded GUS proteins clustered into six unique structural categories. We assign their taxonomy, assess cellular localization, reveal the inter-individual variability within the 139 individuals sampled, and discover 112 novel microbial GUS enzymes. A representative in vitro panel of the most common GUS proteins by read abundances highlights structural and functional variabilities within the family, including their differential processing of smaller glucuronides and larger carbohydrates. These data provide a sequencing-to-molecular roadmap for examining microbiome-encoded enzymes essential to human health.

A metagenomic ß-glucuronidase uncovers a core adaptive function of the human intestinal microbiome.

In the human gastrointestinal tract, bacterial ß-D-glucuronidases (BG; E.C. 3.2.1.31) are involved both in xenobiotic metabolism and in some of the beneficial effects of dietary compounds. Despite their biological significance, investigations are hampered by the fact that only a few BGs have so far been studied. A functional metagenomic approach was therefore performed on intestinal metagenomic libraries using chromogenic glucuronides as probes. Using this strategy, 19 positive metagenomic clones were identified but only one exhibited strong ß-D-glucuronidase activity when subcloned into an expression vector. The cloned gene encoded a ß-D-glucuronidase (called H11G11-BG) that had distant amino acid sequence homologies and an additional C terminus domain compared with known ß-D-glucuronidases. Fifteen homologs were identified in public bacterial genome databases (38-57% identity with H11G11-BG) in the Firmicutes phylum. The genomes identified derived from strains from Ruminococcaceae, Lachnospiraceae, and Clostridiaceae. The genetic context diversity, with closely related symporters and gene duplication, argued for functional diversity and contribution to adaptive mechanisms. In contrast to the previously known ß-D-glucuronidases, this previously undescribed type was present in the published microbiome of each healthy adult/child investigated (n = 11) and was specific to the human gut ecosystem. In conclusion, our functional metagenomic approach revealed a class of BGs that may be part of a functional core specifically evolved to adapt to the human gut environment with major health implications. We propose consensus motifs for this unique Firmicutes ß-D-glucuronidase subfamily and for the glycosyl hydrolase family 2.

Adaptive evolution of heparanase in hypoxia-tolerant Spalax: gene cloning and identification of a unique splice variant.

Heparan sulfate (HS) side chains of HS proteoglycans bind to and assemble extracellular matrix proteins and play important roles in cell-cell and cell-extracellular matrix interactions. HS chains bind a multitude of bioactive molecules and thereby function in the control of multiple normal and pathological processes. Enzymatic degradation of HS by heparanase, a mammalian endoglycosidase, affects the integrity and functional state of tissues and is involved in, among other processes, inflammation, angiogenesis, and cancer metastasis. Here, we report the cloning of heparanase from four Israeli species of the blind subterranean mole rat (Spalax ehrenbergi superspecies), 85% homologous to the human enzyme. Unlike its limited expression in human tissues, heparanase is highly expressed in diverse Spalax tissues. Moreover, we have identified a unique splice variant of the Spalax enzyme lacking 16 aa encoded by exon 7. This deletion resulted in a major defect in trafficking and processing of the heparanase protein, leading to a loss of its enzymatic activity. Interspecies variation was noted in the sequence and in the expression of the splice variant of the heparanase gene in blind mole rats living under different ecogeographical stresses, indicating a possible role in adaptation to stress in Spalax evolution.

A functional screen identifies lateral transfer of beta-glucuronidase (gus) from bacteria to fungi.

Lateral gene transfer (LGT) from prokaryotes to microbial eukaryotes is usually detected by chance through genome-sequencing projects. Here, we explore a different, hypothesis-driven approach. We show that the fitness advantage associated with the transferred gene, typically invoked only in retrospect, can be used to design a functional screen capable of identifying postulated LGT cases. We hypothesized that beta-glucuronidase (gus) genes may be prone to LGT from bacteria to fungi (thought to lack gus) because this would enable fungi to utilize glucuronides in vertebrate urine as a carbon source. Using an enrichment procedure based on a glucose-releasing glucuronide analog (cellobiouronic acid), we isolated two gus(+) ascomycete fungi from soils (Penicillium canescens and Scopulariopsis sp.). A phylogenetic analysis suggested that their gus genes, as well as the gus genes identified in genomic sequences of the ascomycetes Aspergillus nidulans and Gibberella zeae, had been introgressed laterally from high-GC gram(+) bacteria. Two such bacteria (Arthrobacter spp.), isolated together with the gus(+) fungi, appeared to be the descendants of a bacterial donor organism from which gus had been transferred to fungi. This scenario was independently supported by similar substrate affinities of the encoded beta-glucuronidases, the absence of introns from fungal gus genes, and the similarity between the signal peptide-encoding 5' extensions of some fungal gus genes and the Arthrobacter sequences upstream of gus. Differences in the sequences of the fungal 5' extensions suggested at least two separate introgression events after the divergence of the two main Euascomycete classes. We suggest that deposition of glucuronides on soils as a result of the colonization of land by vertebrates may have favored LGT of gus from bacteria to fungi in soils.

Comparative amino acid sequence analysis of Thermotoga maritima beta-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between beta-glucosidases of the BGA family and other families of beta-1,4-glycosyl hydrolases.

The primary structure of the bglA gene region encoding a beta-glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51,545 Da. The T. maritima beta-glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15-20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity beta-glucosidase and as a member of the beta-glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family beta-glucosidases, a beta-xylosidase, beta-1,4-glycanases of the enzyme family F (mostly xylanases), and other families of beta-1,4-glycosyl hydrolases. This result indicates that BGA beta-glucosidases may comprise one enzyme family within a large 'enzyme order' of retaining beta-glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.