1-Deoxymannojirimycin hydrochloride

Hiding inside? Intracellular expression of non-glycosylated c-kit protein in cardiac progenitor cells.[Pubmed:27161312]

Stem Cell Res. 2016 May;16(3):795-806.

Cardiac progenitor cells including c-kit(+) cells and cardiosphere-derived cells (CDCs) play important roles in cardiac repair and regeneration. CDCs were reported to contain only small subpopulations of c-kit(+) cells and recent publications suggested that depletion of the c-kit(+) subpopulation of cells has no effect on regenerative properties of CDCs. However, our current study showed that the vast majority of CDCs from murine heart actually express c-kit, albeit, in an intracellular and non-glycosylated form. Immunostaining and flow cytometry showed that the fluorescent signal indicative of c-kit immunostaining significantly increased when cell membranes were permeabilized. Western blots further demonstrated that glycosylation of c-kit was increased during endothelial differentiation in a time dependent manner. Glycosylation inhibition by 1-Deoxymannojirimycin hydrochloride (1-DMM) blocked c-kit glycosylation and reduced expression of endothelial cell markers such as Flk-1 and CD31 during differentiation. Pretreatment of these cells with a c-kit kinase inhibitor (imatinib mesylate) also attenuated Flk-1 and CD31 expression. These results suggest that c-kit glycosylation and its kinase activity are likely needed for these cells to differentiate into an endothelial lineage. In vivo, we found that intracellular c-kit expressing cells are located in the wall of cardiac blood vessels in mice subjected to myocardial infarction. In summary, our work demonstrated for the first time that c-kit is not only expressed in CDCs but may also directly participate in CDC differentiation into an endothelial lineage.

The use of 1-deoxymannojirimycin to evaluate the role of various alpha-mannosidases in oligosaccharide processing in intact cells.[Pubmed:2937779]

J Biol Chem. 1986 Apr 5;261(10):4766-74.

The mannose analogue, 1-deoxymannojirimycin, which inhibits Golgi alpha-mannosidase I but not endoplasmic reticulum (ER) alpha-mannosidase has been used to determine the role of the ER alpha-mannosidase in the processing of the asparagine-linked oligosaccharides on glycoproteins in intact cells. In the absence of the inhibitor, the predominant oligosaccharide structures found on the ER glycoprotein 3-hydroxy-3-methylglutaryl-CoA reductase in UT-1 cells are single isomers of Man6GlcNAc and Man8GlcNAc. In the presence of 150 microM 1-deoxymannojirimycin, the Man8GlcNAc2 isomer accumulates indicating that the 1-deoxymannojirimycin-resistant ER alpha-mannosidase is responsible for the conversion of Man9GlcNAc2 to Man8GlcNAc2 on reductase. The processing of Man8GlcNAc2 to Man6GlcNAc2, however, must be attributed to a 1-deoxymannojirimycin-sensitive alpha-mannosidase. When cells were radiolabeled with [2-(3)H]mannose for 15 h in the presence of 1-deoxymannojirimycin and then further incubated for 3 h in nonradioactive medium without inhibitor, the Man8GlcNAc2 oligosaccharides which accumulated during the labeling period were partially trimmed to Man6GlcNAc. This finding suggests that a second alpha-mannosidase, sensitive to 1-deoxymannojirimycin, resides in the crystalloid ER and is responsible for trimming the reductase oligosaccharide chain from Man8GlcNAc2 to Man6GlcNAc2. To determine if ER alpha-mannosidase is responsible for trimming the oligosaccharides of all glycoproteins from Man9GlcNAc to Man8GlcNAc, the total asparagine-linked oligosaccharides of rat hepatocytes labeled with [2-(3)H]mannose in the presence or absence of 1.0 mM 1-deoxymannojirimycin were examined. the inhibitor prevented the formation of complex oligosaccharides and caused a 30-fold increase in the amount of Man9GlcNAc2 and a 13-fold increase in the amount of Man8GlcNAc2 present on secreted glycoproteins. This result suggests that only one-third of the secreted glycoproteins is initially processed by ER alpha-mannosidase, and two-thirds are processed by Golgi alpha-mannosidase I or another 1-deoxymannojirimycin-sensitive alpha-mannosidase. The inhibitor caused only a 2.6-fold increase in the amount of Man9GlcNAc2 on cellular glycoproteins suggesting that a higher proportion of these glycoproteins are initially processed by the ER alpha-mannosidase. We conclude that some, but not all, hepatocyte glycoproteins are substrates for ER alpha-mannosidase which catalyzes the removal of a specific mannose residue from Man9GlcNAc2 to form a single isomer of Man8GlcNAc2.

The effect of 1-deoxymannojirimycin on rat liver alpha-mannosidases.[Pubmed:6239623]

Biochem Biophys Res Commun. 1984 Nov 30;125(1):324-31.

The mannose analogue, 1-deoxymannojirimycin, has been tested for its effect on five alpha-mannosidase activities present in rat liver and shown to be a specific inhibitor of Golgi alpha-mannosidase I at low mumolar concentrations. Golgi alpha-mannosidases I and II were assayed in a highly purified Golgi membrane preparation. Endoplasmic reticulum alpha-mannosidase activity was measured in a rough endoplasmic reticulum detergent extract. A purified soluble alpha-mannosidase activity which we believe is derived from the endoplasmic reticulum during tissue homogenization was also tested. And finally, the lysosomal or acidic alpha-mannosidase was measured in a postnuclear supernatant fraction obtained from rat liver. The results presented here show that 1-deoxymannojirimycin inhibits only Golgi alpha-mannosidase I, which is consistent with its effect on oligosaccharide processing in vivo (Fuhrmann et al. Nature 1984 307:755-758).

Novel mannosidase inhibitor blocking conversion of high mannose to complex oligosaccharides.[Pubmed:6230538]

Nature. 1984 Feb 23-29;307(5953):755-8.

Many secretory and membrane proteins are glycoproteins carrying asparagine-linked (N-linked) oligosaccharides. There are two types of N-linked glycans, referred to as high-mannose and complex type, respectively. Biosynthesis of N-linked glycans of the complex type proceeds via a high-mannose intermediate. After the initial transfer of a high-mannose oligosaccharide with the composition (Glc)3(Man)9(GlcNAc)2 from a lipid carrier to the nascent polypeptide chain, trimming reactions take place. Trimming glucosidases remove the glucose residues quantitatively and mannosidases IA/B and II can remove all but three mannose residues. After trimming, terminal sugars such as N-acetylglucosamine, galactose, sialic acid and fucose may be added and result in the conversion to a glycan of the complex type. Because suitable inhibitors were lacking, it was difficult to assess the importance of the trimming reactions for proper intracellular traffic, modification reactions other than the addition of terminal sugars, or as regulatory steps in glycoprotein processing. Here we describe the action of 1-deoxymannojirimycin (1,5-dideoxy-1,5-imino-D-mannitol, dMM; Fig. 1) on the biosynthesis of IgM and IgD. dMM is the mannose analogue of 1-deoxynojirimycin (dNM; Fig. 1), itself a glucosidase inhibitor. We present evidence that dMM is a mannosidase inhibitor. In vivo dMM inhibits the equivalent of the mannosidase IA/B activities and blocks conversion of high-mannose to complex oligosaccharides. It is the first such inhibitor to be reported. Interference with the biosynthetic pathway of N-linked glycans could prove to be a powerful way to manipulate carbohydrate structure in vivo.