Sialidase (neuraminidase, EC 3.2.1.18) catalyses the hydrolysis of terminal sialic acid residues of glyconjugates. Sialidase has been well studied in viruses and bacteria where it destroys the sialic acid-containing receptors at the surface of host cells, and mobilizes bacterial nutrients. In mammals, three types of sialidases, lysosomal, plasma membrane and cytosolic, have been described. For lysosomal sialidase in humans, the primary genetic deficiency results in an autosomal recessive disease, sialidosis, associated with tissue accumulation and urinary excretion of sialylated oligosaccharides and glycolipids. Sialidosis includes two main clinical variants: late-onset, sialidosis type I, characterized by bilateral macular cherry-red spots and myoclonus, and infantile-onset, sialidosis type II, characterized by skeletal dysplasia, mental retardation and hepatosplenomegaly. We report the identification of human lysosomal sialidase cDNA, its cloning, sequencing and expression. Examination of six sialidosis patients revealed three mutations, one frameshift insertion and two missense. We mapped the lysosomal sialidase gene to human chromosome 6 (6p21.3), which is consistent with the previous chromosomal assignment of this gene in proximity to the HLA locus.
Sialidosis is an autosomal recessive disease caused by the genetic deficiency of lysosomal sialidase, which catalyzes the hydrolysis of sialoglycoconjugates. The disease is associated with progressive impaired vision, macular cherry-red spots and myoclonus (sialidosis type I) or with skeletal dysplasia, Hurler-like phenotype, dysostosis multiplex, mental retardation and hepatosplenomegaly (sialidosis type II). We have analyzed the genomic DNA from nine sialidosis patients of multiple ethnic origin in order to find mutations responsible for the enzyme deficiency. The activity of the identified variants was studied by transgenic expression. One patient had a frameshift mutation (G623delG deletion), which introduced a stop codon, truncating 113 amino acids. All others had missense mutations: G679G-->A (Gly227Arg), C893C-->T (Ala298Val), G203G-->T (Gly68Val), A544A-->G (Ser182Gly) C808C-->T (Leu270Phe) and G982G-->A (Gly328Ser). We have modeled the three-dimensional structure of sialidase based on the atomic coordinates of the homologous bacterial sialidases, located the positions of mutations and estimated their potential effect. This analysis showed that five mutations are clustered in one region on the surface of the sialidase molecule. These mutations dramatically reduce the enzyme activity and cause a rapid intralysosomal degradation of the expressed protein. We hypothesize that this region may be involved in the interface of sialidase binding with lysosomal cathepsin A and/or beta-galactosidase in their high-molecular-weight complex required for the expression of sialidase activity in the lysosome.
We describe the identification, cDNA cloning, and biochemical characterization of a new human blood plasma glutamate carboxypeptidase (PGCP). PGCP was co-purified from human placenta with lysosomal carboxypeptidase, cathepsin A, lysosomal endopeptidase, cathepsin D, and a ␥-interferon-inducible protein, IP-30, using an affinity chromatography on a Phe-Leu-agarose column. A PGCP cDNA was obtained as an expressed sequence tag clone and completed at 5-end by rapid amplification of cDNA ends polymerase chain reaction. The cDNA contained a 1623-base pair open reading frame predicting a 541-amino acid protein, with five putative Asn glycosylation sites and a 21-residue signal peptide. PGCP showed significant amino acid sequence homology to several cocatalytic metallopeptidases including a glutamate carboxypeptidase II also known as N-acetyl-aspartyl-␣-glutamate carboxypeptidase or as prostate-specific membrane antigen and expressed glutamate carboxypeptidase activity. Expression of the PGCP cDNA in COS-1 cells, followed by Western blotting and metabolic labeling showed that PGCP is synthesized as a 62-kDa precursor, which is processed to a 56-kDa mature form containing two Asn-linked oligosaccharide chains. The mature form of PGCP was secreted into the culture medium, which is consistent with its intracellular localization in secretion granules. In humans, PGCP is found principally in blood plasma, suggesting a potential role in the metabolism of secreted peptides.Cellular and secreted carboxypeptidases are important in the generation, processing, and inactivation of different vertebrate neuropeptides (for recent reviews, see Refs.
Porphobilinogen deaminase is the third enzyme in the heme biosynthetic pathway. hem3 mutants in Saccharomyces cerevisiae are deficient in porphobilinogen deaminase activity. We have isolated the HEM3 gene by complementation of the heme auxotrophy of a hem3 mutant. Sequence analysis reveals an open reading frame of 981 nucleotides. The derived amino acid sequence of the protein encoded by HEM3 shows extensive homology to the reported sequences for porphobilinogen deaminase from a number of other sources, indicating that HEM3 is the structural gene for porphobilinogen deaminase. Earlier reports have suggested that expression of HEM3 is induced by porphobilinogen, the substrate of the encoded enzyme. We have investigated the transcription of HEM3 and have found that it is not affected by the ability of the cell to make porphobilinogen or heme. However, we have found that HAP2 and HAP3 gene products are involved in the expression of HEM3. An important element required for expression of HEM3 has been localized to a small region that contains a sequence homologous to the HAP2-3-4 binding sites of several genes including HEM1. These findings suggest that HEM3 expression is regulated in the same manner as that of HEM1 which encodes the first enzyme of the heme biosynthetic pathway.
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AbstractA large proportion of Petroleum Development Oman (PDO) future production resides in fractured reservoirs. In order to support the development of these volumes, a strong element of fracture characterisation and modelling has been included within a number of subsurface studies. The key enabler for these studies is the software technology, SVS (Simple Visualisation Software), developed by the Carbonate Development Team (CDT), in Shell EP-Research. PDO has not only taken a lead role in software implementation but is also steering the ongoing development of SVS according to the needs of active field studies. Currently, SVS is applied to the three themes of Oman's fractured reservoirs: (1) slightly fractured containing light oil (2) medium/highly fractured containing light oil and (3) highly fractured containing medium-heavy oil. The key pillar of the SVS workflow is a detailed fracture characterisation which leads to the elaboration of a series of conceptual models which capture the range of the subsurface uncertainties. Once the conceptual models have been developed, these can be transformed into discrete fracture models with attached attributes (such as permeability anisotropy, fracture spacing etc). These models maybe transformed into reservoir simulation properties as per study requirements. This complementary paper to Rawnsley et al 2004 1 has for main objective to illustrate the SVS workflow with particular emphasis on the borehole image analysis and the use of a web based sandbox model database, to help constrain the fault geometries and the structural understanding of the fields.
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