Protein N-glycosylation in trypanosomatids. A pathway with odd enzymes at both ends

This review deals with the pathway leading to the synthesis of asparagine-linked oligosaccharides in trypanosomatid protozoa. Special emphasis is put on steps differing from those occurring in mammalian cells

Characterization of solubilized GlcAT-1 (UDP-GlcA: nLcOse4Cer beta 1-3 glucuronyltransferase) activity from embryonic chicken brain and its inhibition by D-erythro-sphingosine.

Glycolipid glucuronyltransferase activity (GlcAT-1) has been solubilized and characterized from 19-day-old embryonic chicken brain Golgi-rich membranes. The enzyme catalyzes the biosynthesis in vitro of GlcA beta 1-3nLcOse4Cer glycolipid using neolactetraosylceramide (nLcOse4Cer, Gal beta 1-4GlcNAc beta 1-3Gal beta-1-4Glc-Cer) as the substrate. The membrane-bound enzyme shows optimum activity in the presence of neutral detergents such as Triton CF-54, Triton DF-12, and Nonidet P-40. Approximately 60% of the enzyme activity can be solubilized from the Golgi membrane by Nonidet P-40. The solubilized GlcAT-1 activity is inhibited by different salts such as NaCl, NaBr, NaI, and NaOAc, but not by sodium fluoride (up to 0.4 M concentration). Desialyzed alpha 1 acid glycoprotein (SA alpha 1AGP) can be used as a substrate for glucuronyltransferase. Competition studies between glycolipid (nLcOse4Cer) and glycoprotein SA alpha 1AGP) substrates show a mixed type of inhibition. Phospholipids, in particular phosphatidylglycerol, stimulate solubilized GlcAT-1 activity, while D-erythro-sphingosine, a metabolite of glycosphingolipids, is inhibitory (50% inhibition at 0.8 mM D-erythro-sph). These results demonstrate that both phospholipid as well as sphingosine might be involved in modulating glucuronyltransferase activity.

Carbohydrate and hydrophobic-carbohydrate recognition sites (CARS and HY-CARS) in solubilized glycosyltransferases.

Six different glycosyltransferases that are active with glycosphingolipid substrates have been purified from Golgi-membranes after solubilization with detergents. It appears that GalT-4(UDP-Gal:GlcNAc-R1 beta 1-4GalT), GalNAcT-2(UDP-Gal:Gal alpha-R2 beta 1-3GalNAcT) and FucT-2(GDP-Fuc:Gal beta GlcNAc-R3 alpha 1-2FucT) are specific for oligosaccharides bound to ceramide or to a protein moiety. These are called CARS (carbohydrate recognition sites) glycosyltransferases (GLTs). On the other hand, GalT-3(UDP-Gal:GM2 beta 1-3GalT), GalNAcT-1(UDP-GalNAc:GM3 beta 1-4GalNAcT) and FucT-3 (GDP-Fuc:LM1 alpha 1-3FucT) recognize both hydrophobic moieties (fatty acid of ceramide) as well as the oligosaccharide chains of the substrates. These GLTs are called HY-CARS (hydrophobic and carbohydrate recognition sites). D-Erythro-sphingosine (100-500 microM) modulates the in vitro activities of these GLTs. Modulation depends on the binding of D-sphingosine to a protein backbone, perhaps on more than one site and beyond transmembrane hydrophobic domains. Control of GLTs by free D-sphingosine was suggested with the concomitant discovery of ceramide glycanase in rabbit mammary tissues. The role of free sphingosine as an in vivo homotropic modulator of glycosyltransferases is becoming apparent.