Neuraminidases

The properties of a neuraminidase in the intestines of young mammals have been investigated.  

The fibroblasts from a patient with mucolipidosis I were found to be severely deficient in an acid neuraminidase. Since normal levels of other glycoside hydrolases were found, the basic metabolic lesion appears to involve a defect in the degradation of compounds containing 5-acetamido-3,5-dideoxy-D-gf/yc ro-D-galacto-nonxxiosonic acid. 

Although the addition of sodium butyrate to HeLa cells in culture increased the synthesis of ganglioside, the levels of sialidase and other glycoside hydrolases were not affected. The sialidase hydrolysed 0, and gangliosides, but not fetuin the enzyme has a pH optimum of 5.0 and a value of 75 pmol 1  

A sensitive radiometric assay for the neuraminidase from calf brain uses a tritium-labelled G, ganglioside as the substrate. After incubation with the enzyme, the substrate and the product ([ Hjganglioside G ) are separated by t.l.c. and their radioactivities measured. 

The relationships between bacterial neuraminidases and alterations in the immunological behaviour of neuraminidase-treated mammalian cells have been reviewed.  

A simple method for preparing commercial neuraminidase (sialidase) free from proteases has been reported.  

The effects of neuraminidase and of anti-neuraminidase sera on the properties of cell surfaces have been investigated. The activities and properties of diploid Wl-38 fibroblasts from embryonic human-lung tissues have been compared with those of other acid glycoside hydrolases from the same source.  

The removal of residues of 5-acetamido-3,5-dideoxy-D- /ycero-D- a/acm-2-nonulosonic acid from particulate glycoproteins of calf brain by endogenous, membrane-bound neuraminidase has been studied.  

Neuraminidase has been found in porcine gastric mucus the enzyme probably originates from bacteria present in mucous secretions, and is considered to affect the balance of gastric glycoproteins.  

The pathology of microbial neuraminidases has been reviewed numerous sources of the enzyme were considered, and diseases associated with neuraminidases were tabulated.  

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Jedrzejas, M. J., Singh, S. Brouillette, W. J. Air, G. M. Luo, M. A. 1995. Strategy for theoretical binding constant, Ki calculation for neuraminidase aromatic inhibitors, designed on the basis of the active site structure of influenza virus neuraminidase. Proteins Struct. Funct. Genet. 23 (1995) 264-277... 

Neuleptil Neuphor Neupramir Neural nets Neural stimulators Neuraminidase... 

Subunit vaccines based on the surface proteins of vims are also being explored. It has been demonstrated that the two major protective antigens are haemagglutinin (HA) and neuraminidase (NA). The genes for these antigens have been cloned and expressed in baculovims in insect cell culture (84). 

Enzymes Degrading Macromolecules. Enzymes that degrade macromolecules such as membrane polysaccharides, stmctural and functional proteins, or nucleic acids, have all shown oncolytic activity. Treatment strategies include the treatment of inoperable tumors with pepsin (1) antitumor activity of carboxypeptidase (44) cytotoxicity of ribonudease (45—47) oncolytic activity of neuraminidase (48—52) therapy with neuraminidase of patients with acute myeloid leukemia (53) antitumor activity of proteases (54) and hyaluronidase treatment in the management of human soHd tumors (55). 

A second example of up-and-down p sheets is the protein neuraminidase from influenza virus. Here the packing of the sheets is different from that in RBP. They do not form a simple barrel but instead six small sheets, each with four P strands, which are arranged like the blades of a six-bladed propeller. Loop regions between the p strands form the active site in the middle of one side of the propeller. Other similar structures are known with different numbers of the same motif arranged like propellers with different numbers of blades such as the G-proteins discussed in Chapter 13. 

The role of the viral neuraminidase, conversely, seems to be to facilitate the release of progeny virions from infected cells by cleaving sialic acid... 

The neuraminidase molecule is a homotetramer made up of four identical polypeptide chains, each of around 470 amino acids the exact number varies depending on the strain of the virus. If influenza virus is treated with the proteolytic enzyme pronase, the head of the neuraminidase, which is soluble, is cleaved off from the stalk projecting from the viral envelope. The soluble head, comprising four subunits of about 400 amino acids each, can be crystallized. 

Folding motifs form a propeller-like structure in neuraminidase... 

Figure S.6 Schematic and topological diagrams of the folding motif in neuraminidase from influenza virus The motif is built up from four antiparallel P strands joined by hairpin loops, an up-and-down open P sheet.

Figure S.7 The subunit structure of the neuraminidase headpiece (residues 84-469) from influenza virus is built up from six similar, consecutive motifs of four up-and-down antiparallel fi strands (Figure 5.6). Each such motif has been called a propeller blade and the whole subunit stmcture a six-blade propeller. The motifs are connected by loop regions from p strand 4 in one motif to p strand 1 in the next motif. The schematic diagram (a) is viewed down an approximate sixfold axis that relates the centers of the motifs. Four such six-blade propeller subunits are present in each complete neuraminidase molecule (see Figure 5.8). In the topological diagram (b) the yellow loop that connects the N-terminal P strand to the first P strand of motif 1 is not to scale. In the folded structure it is about the same length as the other loops that connect the motifs. (Adapted from J. Varghese et al.. Nature 303 35-40, 1983.)...

Figure S.8 Schematic view down the fourfold axis of the tetrameric molecule of neuraminidase as It appeared on the cover of Nature, May 5, 1983.

Figure 5.9 The six four-stranded motifs in a single subunit of neuraminidase form the six blades of a propeller-like structure. A schematic diagram of the subunit structure shows the propeller viewed from its side (a). An idealized propeller structure viewed from the side to highlight the position of the active site is shown in (b). The loop regions that connect the motifs (red in b) in combination with the loops that connect strands 2 and 3 within the motifs (green in b) form a wide funnel-shaped active site pocket, [(a) Adapted from P. Colman et ah, Nature 326 358-363, 1987.]...

We have already discussed one envelope protein of influenza virus, neuraminidase, as an example of an up-and-down antiparallel p motif. In the second envelope protein, hemagglutinin, one domain of the polypeptide chain is folded into a jelly roll motif. We shall now look at some other features of hemagglutinin that are important for its biological function. 

Progeny vims particles then bud from patches of the infected cell s plasma membrane that contain both the viral hemagglutinin and neuraminidase. The viral envelopes therefore contain both viral membrane proteins but no cellular membrane proteins. 

The second protein in the membrane of influenza vims, neuraminidase, does not belong to any of these three groups of barrel structures. Instead, it forms a propeller-like structure of 24 p strands, arranged in six similar motifs that form the six blades of the propeller. Each motif is a p sheet of 4 up-and-down-connected p strands. The enzyme active site is formed by loop regions on one side of the propeller. 

Colman, P.M., Varghese, J.N., Laver, W.G. Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature 303 41-44, 1983. 

Colman, P.M., et al. Three-dimensional structure of a complex of antibody with influenza virus neuraminidase. Nature 326 358-363, 1987. 

The Diels-Alder reaction of nitroalkenes with Danishefsky s dienes is applied to synthesis of truncated carbocyclic analogues of a potent neuraminidase inhibitor 4-guanidino-NemAc en fsee Scheme 8.5. Carbocyclic analogs are found to retain interesting levels of antiviral activity comparable to those shovm by their oxygen-containing compounds in Scheme 8.5. 

The influenza virus inhibitors, zanamivir, and oseltamivir, act outside the cell after virus particles have been formed. The dtugs have been designed to fit into the active site of the viral envelope enzyme neuraminidase, which is required to cleave sialic acid off the surface of the producing cells. When its activity is blocked, new virus particles stay attached to the cell surface through binding of the virus protein hemagglutinin to sialic acid and are prevented from spreading to other cells. 

Neuraminidase inhibitors are the major class of drugs to treat or to prevent the infection with influenza viruses. Currently, two neuraminidase inhibitors are available, zanamivir and oseltamivir, which block the release of new influenza vims from infected host cells and thereby stop the spread of infection. The enzyme neuraminidase is a surface glycoprotein present on all influenza viruses. There are nine influenza neuraminidase sub-types known of which subtypes N1 and N2 appear to be the most important ones. Neuraminidase inhibitors are effective against all neuraminidase subtypes. The activity of the neuraminidase is required for the newly... 

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