(1S,2R,8R,8aR)-1,2,8-trihydroxyoctahydroindolizine (swainsonine), an α-mannosidase inhibitor from Rhizoctonia leguminicola

“…This oligosaccharide (NANAGalGIcNAcMansGIcNAc) was treated with sialidase and fl-D-galactosidase as described (38), and the resulting oligosaccharide (GIcNAcMansGIcNAc) was purified by high resolution gel filtration on a column of Bio-Gel P-4 (42). Swainsoninc isolated from Rhizoctonia leguminicola (32) was provided by Dr. H. P. Broquist of this university, l-Deoxymannojirimycin was from Boehringer Mannheim Diagnostics, Inc., Houston, TX. Highly specific polyclonal antibody (IgG fraction) against rat liver Golgi mannosidase IA was prepared as described (41).…”

Section: Methodsmentioning

confidence: 99%

Novel alpha-D-mannosidase of rat sperm plasma membranes: characterization and potential role in sperm-egg interactions.

Tulsiani

Skudlarek

Orgebin‐Crist

1989

The Journal of Cell Biology

130

100

View full text Add to dashboard Cite

Abstract. During the course of a study of glycoprotein processing mannosidases in the rat epididymis, we have made an intriguing discovery regarding the presence of a novel ot-o-mannosidase on the rat sperm plasma membranes. Unlike the sperm acrosomal "acid" mannosidase which has a pH optimum of 4.4, the newly discovered Ot-D-mannosidase has a pH optimum of 6.2, and 6.5 when assayed in sperm plasma membranes and intact spermatozoa, respectively. In addition, the two enzymes show different substrate specificity. The acrosomal Ot-D-mannosidase is active mainly towards synthetic substrate, p-nitrophenyl a-Dmannopyranoside, whereas the sperm plasma membrane O~-D-mannosidase shows activity mainly towards mannose-containing oligosaccharides. Evidence is presented which suggest that the sperm plasma membrane Ot-D-mannosidase is different from several processing mannosidases previously characterized from the rat liver.The newly discovered a-D-mannosidase appears to be an intrinsic plasma membrane component, since washing of the purified membranes with buffered 0.4 M NaCI did not release the enzyme in soluble form. The enzyme requires nonionic detergent (Triton X-100) for complete solubilization. The enzyme is activated by Co 2+ and Mn 2÷. However, Cu 2÷ and Zn 2÷ are potent inhibitors of the sperm plasma membrane Ot-D-mannosidase. At a concentration of 0.1 mM, these divalent cations caused nearly complete inactivation of the sperm enzyme. In addition methyl-c~-D-mannoside, methyl-ot-D-glucoside, mannose, 2-deoxy-D-glucose, and D-mannosamine are inhibitors of the sperm surface a-D-mannosidase. The physiological role of the newly discovered enzyme is not yet known. Several published reports in three species, including the rat, suggest that the sperm surface c~-o-mannosidase may have a role in binding to mannose-containing saccharides presumably present on the zona pellucida.I X is generally accepted that one step in the fertilization process requires interaction between complementary molecules present on the surface of the spermatozoon and the zona pellucida. The chemical nature of these complementary recognition sites is poorly understood, although there is growing evidence that carbohydrate moieties on surface membrane glycoconjugates are involved in these interactions (1, 34).In mammals, several sperm proteins have been suggested to bind to zona pellucida (28). Some of these macromolecules have enzymatic activity and are thought to form a stable enzyme-substrate complex by binding to the oligosaccharide units present on the zona pellucida glycoproteins. In mice, galactosyltransferase present on the head region of the spermatozoa mediates sperm-egg binding by interacting with its substrate on the zona pellucida (23). There is also evidence that trypsin-like protease present on spermatozoa of mouse (4, 31) initiates sperm-egg binding. Mouse spermatozoa also contain sialyltransferase (13) and fucosyltransferase (30). The latter enzyme has been suggested to be involved in some aspect of sperm-egg recognition (Apter, E M., ...

show abstract

“…(Molyneux & James, 1982) and in the fungus Rhizoctonia leguminicola (Schneider et al, 1982). It is a potent inhibitor of a-D-mannosidase activity (Dorling et al, 1980).…”

mentioning

confidence: 99%

The storage products in genetic and swainsonine-induced human mannosidosis

Bello¹,

Dorling²,

Winchester³

1983

Biochemical Journal

View full text Add to dashboard Cite

Swainsonine induces the accumulation of mannose-rich oligosaccharides in human fibroblasts. The composition of the storage products shows that swainsonine completely inhibits lysosomal a-D-mannosidase and alters processing of glycoproteins by inhibiting Golgi a-D-mannosidase II. Comparison of the storage products in genetic and swainsonine-induced mannosidosis suggests that human fibroblasts contain a lysosomal a-D-mannosidase that is unaffected in genetic mannosidosis.The 1,2,8-trihydroxylated indolizidine alkaloid swainsonine occurs in plants of the genus Swainsona (Colegate et al., 1979), in certain species of locoweeds (Astragalus and Oxytropis spp.) (Molyneux & James, 1982) and in the fungus Rhizoctonia leguminicola (Schneider et al., 1982). It is a potent inhibitor of a-D-mannosidase activity (Dorling et al., 1980). Prolonged ingestion of swainsonine by animals leads to a neurological disorder that is a phenocopy of the genetically induced lysosomal storage disease mannosidosis (Dorling et al., 1978).Mannosidosis is characterized by accumulation in cells, and excretion in the urine, of mannose-rich oligosaccharides resulting from a deficiency of lysosomal a-D-mannosidase (Jolly et al., 1981). Swainsonine is a lysosomotropic compound and accumulates rapidly in lysosomes of normal human fibroblasts in culture to produce inhibition of intracellular lysosomal a-D-mannosidase (Chotai et al., 1983). It is assumed that this is its mode of action in vivo. Swainsonine also affects one (Elbein et al., 1981(Elbein et al., , 1982Gross et al., 1983). In the present paper we show that swainsonine induces the storage of mannose-rich oligosaccharides and alters the processing of glycoproteins in human fibroblasts in culture. The storage products in swainsonineinduced and genetic human mannosidosis are compared. Methods ChemicalsAll reagents were of analytical grade and were obtained from BDH or Sigma, both of Poole, Dorset, U.K. Swainsonine was isolated from the plant Swainsona canescens (Benth.) A. Lee by the method of Colegate et al. (1979) Normal human fibroblasts and fibroblasts from a child with mannosidosis were grown as described previously (Burditt et al., 1978). To investigate the effect of swainsonine on the metabolism of glycoproteins, the cells were incubated in culture medium containing 100 pM-swainsonine for the required length of time. The reversal of swainsonine-induced effects was followed by replacing the swainsoninecontaining medium after the required length of time with normal medium. The monolayers of cells were

show abstract

(1S,2R,8R,8aR)-1,2,8-trihydroxyoctahydroindolizine (swainsonine), an α-mannosidase inhibitor from Rhizoctonia leguminicola

Cited by 173 publications

References 14 publications

Asymmetric Synthesis of ( - )-Swainsonine

Asymmetric Synthesis of ( - )-Swainsonine

Novel alpha-D-mannosidase of rat sperm plasma membranes: characterization and potential role in sperm-egg interactions.

The storage products in genetic and swainsonine-induced human mannosidosis

Contact Info

Product

Resources

About