Evaluation of the role of rat liver Golgi endo-alpha-D-mannosidase in processing N-linked oligosaccharides.

Golgi membranes from rat liver have been shown to contain an endo-alpha-D-mannosidase which can convert Glc1Man9GlcNAc to Man8GlcNAc with the release of Glc alpha 1----3Man (Lubas, W. A., and Spiro, R. G. (1987) J. Biol. Chem. 262, 3775-3781). We now report that this enzyme has the capacity to cleave the alpha 1----2 linkage between the glucose-substituted mannose residue and the remainder of the polymannose branch in a wide range of oligosaccharides (Glc3Man9GlcNAc to Glc1Man4GlcNAc) as well as glycopeptides and oligosaccharide-lipids. Whereas the tri- and diglucosylated species (Glc3Man9GlcNAc and Glc2Man9GlcNAc), which yielded Glc3Man and Glc2Man, respectively, were processed more slowly than Glc1Man9GlcNAc, the monoglucosylated components with truncated mannose chains (Glc1Man8GlcNAc to Glc1Man4GlcNAc) were trimmed at an increased rate which was inversely related to the number of mannose residues present. The endomannosidase was not inhibited by a number of agents which are known to interfere with N-linked oligosaccharide processing by exoglycosidases, including 1-deoxynojirimycin, castanospermine, bromoconduritol, 1-deoxymannojirimycin, swainsonine, and EDTA. However, Tris and other buffers containing primary hydroxyl groups substantially decreased its activity. After Triton solubilization, the endomannosidase was observed to be bound to immobilized wheat germ agglutinin, indicating the presence of a type of carbohydrate unit consistent with Golgi localization of the enzyme. The Man8GlcNAc isomer produced by endomannosidase action was found to be processed by Golgi enzymes through a different sequence of intermediates than the rough endoplasmic reticulum-generated Man8GlcNAc variant, in which the terminal mannose of the middle branch is absent. Whereas the latter oligosaccharide is converted to Man5GlcNAc via Man7GlcNAc and Man6GlcNAc at an even rate, the processing of the endomannosidase-derived Man8GlcNAc stalls at the Man6GlcNAc stage due to the apparent resistance to Golgi mannosidase I of the alpha 1,2-linked mannose of the middle branch. The results of our study suggest that the Golgi endomannosidase takes part in a processing route for N-linked oligosaccharides which have retained glucose beyond the rough endoplasmic reticulum; the distinctive nature of this pathway may influence the ultimate structure of the resulting carbohydrate units.

Control of N-linked carbohydrate unit synthesis in thyroid endoplasmic reticulum by membrane organization and dolichyl phosphate availability.

Thyroid rough endoplasmic reticulum (ER) has been shown to contain a highly organized multienzyme system capable of carrying out the N-glycosylation of newly synthesized proteins. These reactions were studied in isolated ER vesicles and found to be controlled to a large extent by the availability of a key substrate, dolichyl phosphate (Dol-P), as well as by the amount of endogenous polypeptide acceptor present. Although in intact vesicles UDP-Glc was utilized in an efficient manner to form Dol-P-Glc and glucosylated oligosaccharide-lipid, after disruption of vesicle integrity, even with low concentrations of Triton X-100, the coupling of Dol-P-Glc formation to lipid-linked oligosaccharide assembly and subsequent N-glycosylation was substantially impaired. Increased incubation temperatures also resulted in a decreased effectiveness of glucose transfer from Dol-P-Glc to lipid-oligosaccharide, presumably because of a decline in the extent of structural organization of the ER membranes. The limited availability of endogenous Dol-P was demonstrated by the pronounced stimulation in Dol-P-Glc formation resulting from the addition of this lipid acceptor to Triton-disrupted ER membranes as well as by its generation in intact vesicles. The latter was accomplished by stimulating recycling of endogenous Dol-P through the addition of a peptide (Tyr-Asn-Leu-Thr-Ser-Val) which is an N-glycosylation substrate. The inhibition of Dol-P-Glc synthesis from UDP-Glc observed in the presence of elevated levels of GDP-Man which could be relieved in Triton-disrupted or intact ER vesicles by the addition or generation, respectively, of Dol-P, is considered to be the result of a competing requirement for Dol-P by the mannosyltransferase. Moreover GTP, by selectively inhibiting the mannosyltransferase, prevented the decrease of Dol-P-Glc formation caused by GDP-Man. Since addition of the acceptor peptide to intact vesicles stimulated Dol-P-P-GlcNAc as well as Dol-P-Glc and Dol-P-Man synthesis it would appear that a pool of Dol-P available in common to all three enzymes responsible for dolichol-linked monosaccharide synthesis exists in the ER membranes.

Effect of phospholipids on thyroid oligosaccharyltransferase activity and orientation. Evaluation of structural determinants for stimulation of N-glycosylation.

Oligosaccharyltransferase solubilized by Nonidet P-40 was found to have a highly specific lipid requirement which is consistent with the lability of the enzyme when removed from its membrane association. Enzyme activity as measured by the N-glycosylation of a hexapeptide acceptor was greatly stimulated and stabilized by phosphatidylcholine (PC) while other naturally occurring phosphoglycerides had minimal effect. The quaternary ammonium group of PC was observed to be involved in the interaction with the enzyme as modification of the choline moiety by removal of methyl groups resulted in a progressive loss of the stimulatory effect (choline greater than N,N-dimethylethanolamine greater than N-monomethylethanolamine greater than ethanolamine) which was reflected primarily in the Vmax rather than the Km values. Evaluation of a number of PC and choline derivatives indicated that the nonpolar domain of the lipid also played an important specifying role. Two hydrophobic chains attached to the phosphoglycerol backbone were found to be essential, and furthermore the length and degree of unsaturation of the fatty acid substituents as well as their position of attachment on the glycerol moiety greatly affected the extent of activation. Since the L-isomer of PC brought about a 3-fold greater stimulation than the D-isomer the interaction of the enzyme with the phospholipid appears to be stereoselective. Upon chromatography of the PC-stabilized enzyme on concanavalin A-agarose almost complete retention occurred at 0.4% Nonidet P-40, while no binding took place at a detergent concentration of 0.075%; this suggested that upon dilution in the presence of PC, the oligosaccharyltransferase was reconstituted into vesicles in an asymmetric fashion with its N-linked carbohydrate located internally. Enzymatic assay of these vesicles demonstrated that the active site of the enzyme was also oriented toward the interior. These studies indicate that the activity as well as the membrane insertion of the oligosaccharyltransferase are to a large measure influenced by its interaction with PC.

Cleavage of dolichyl pyrophosphoryl oligosaccharides by endo-beta-N-acetylglucosaminidase H: comparison of enzymatic and acid hydrolysis techniques for saccharide release.

Endo-beta-N-acetylglucosaminidase H (endo H) was found to bring about the complete hydrolysis of dolichyl pyrophosphoryl oligosaccharides. Both glycosylated and unglucosylated polymannose oligosaccharides were released by the enzyme through cleavage of the di-N-acetylchitobiose sequence. The action of the endo H on the oligosaccharide-lipids was facilitated by the inclusion of Triton X-100 (maximal stimulation at concentrations greater than 0.03%) or small amounts of a variety of other detergents; however, sodium dodecyl sulfate (0.1%) was strongly inhibitory. Although incubations were routinely carried out at pH 5.2, the enzyme was noted to be equally effective at pH 6.5 and to retain 75% of its activity toward oligosaccharide-lipid at pH 7.4. While these results broaden the known specificity of the endo H for the aglycon moiety, it was observed that even under optimal conditions the rate of hydrolysis of lipid-linked Glc3Man9GlcNAc2 was substantially slower than that of the same oligosaccharide attached to asparagine in a peptide sequence. The use of endo H, an enzyme which can be obtained free of exoglycosidases, appears to have a number of advantages over mild acid hydrolysis as a tool for cleaving oligosaccharide-lipids. It was found that the latter procedure causes a small but detectable degradation of the sugar chains and, when carried out in the presence of methanol, leads to the release of about 10% of the oligosaccharide as its beta-methyl glycoside. Furthermore, the oligosaccharides released by the endo H can be directly compared to those liberated by this enzyme from glycoproteins; this may prove to be useful in metabolic studies dealing with oligosaccharide-lipid assembly and their involvement in the N-glycosylation of proteins.

Lens epithelial cell adhesion to lens capsule: a model system for cell-basement membrane interaction.

Adhesion of calf lens epithelial cells to lens capsule, their natural basement membrane was found to be considerably more rapid than either to plastic or to type I or type IV collagen coated surfaces. No polarity of the basement membrane was observed as the cells were able to attach to either side of the anterior or posterior lens capsule; a prerequisite for adhesion to the lenticular side of the anterior capsule was the prior removal of its epithelial cell layer. The attachment was energy-dependent and required calcium and magnesium ions, but was not enhanced by the presence of serum. Neither exogenous fibronectin nor laminin was able to stimulate attachment or spreading of lens cells to the capsule even when the cells had been treated with cycloheximide. Since rapid adhesion and spreading takes place in this lens cell-lens capsule system without requirement of exogenous macromolecules, it provides a favorable model for investigating the determinants in epithelial cell-basement membrane interactions.

Characterization of a thyroid sulfotransferase responsible for the 3-O-sulfation of terminal beta-D-galactosyl residues in N-linked carbohydrate units.

Calf thyroid microsomes were found to contain an enzyme which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phospho[35S]sulfate (PAPS) to C-3 of terminal galactose residues in beta 1----4 linkage to GlcNAc. This sulfotransferase is believed to be involved in the biosynthesis of the recently described Gal(3-SO4) capping groups present in the N-linked oligosaccharides of thyroglobulin (Spiro, R.G., and Bhoyroo, V. D. (1988) J. Biol. Chem. 263, 14351-14358). Assays with various native and modified glycopeptides indicated that the enzyme acted optimally on complex-type carbohydrate units in which beta-linked Gal has been uncovered by desulfation or brought into a terminal position by removal of sialyl and/or alpha-galactosyl residues. With fetuin asialoglycopeptides as acceptors (Km = 0.1 mM) the transfer of sulfate from PAPS (Km = 6.3 microM) had a pH optimum of approximately 7.0, required Mn2+ ions (10-50 mM) and was markedly stimulated by Triton X-100 (0.1%) and ATP (2 mM). The same enzyme apparently sulfated free N-acetyllactosamine (LacNAc; Km = 0.69 mM) and its ethyl glycoside, indicating that it had no absolute requirement for a peptide recognition site. Studies with a number of disaccharides related to LacNAc provided information relating to the specifying role of the beta 1----4 galactosyl linkage and the configuration at C-2 of the sugar to which it is attached. Hydrazine-nitrous acid-NaBH4 treatment of the 35S-labeled products from sulfotransferase action on asialoglycopeptides as well as on the ethyl glycoside of LacNAc yielded the same disaccharide, Gal(3-SO4) beta 1----4 anhydromannitol, as is obtained from a similar treatment of thyroglobulin. Subcellular distribution studies indicated that the PAPS:galactose 3-O-sulfotransferase is located in the Golgi compartment which is consistent with the late occurrence of the requisite beta-galactosylation step. It is proposed that in certain tissues the ultimate nature of the capping groups attached to glycoproteins containing terminal Gal beta 1----4GlcNAc sequences could be the result of a competition between this 3-O-sulfotransferase and sialyl- and/or alpha-galactosyltransferases.