Rat liver chitobiase: purification, properties, and role in the lysosomal degradation of Asn-linked glycoproteins

Chitobiase, the lysosomal glycosidase responsible for splitting the GlcNAc beta-D-(1-4)GlcNAc moiety in Asn-linked glycoproteins, was purified over 600-fold from frozen rat livers utilizing an assay with di-N-acetylchitobiose as the substrate. The final preparation showed a major polypeptide of Mr 43,000 (sodium dodecylsulfate-polyacrylamide gel electrophoresis) that was determined to be the chitobiase by an immunological method. The purified chitobiase also hydrolyzed tri- and tetrasaccharides of chitin, which like di-N-acetylchitobiose were not substrates if first reduced by NaBH4. The initial products formed during hydrolysis of the tetrasaccharide were trisaccharide and GlcNAc. These results imply that chitobiase is a "reducing-end exohexosaminidase" which cleaves single GlcNAc units only from the reducing end of oligosaccharides. Fucose, typically found linked to the reducing-end GlcNAc in complex oligosaccharide chains, was found to block this reaction. Additional substrates that were hydrolyzed included GlcNAc beta-D-(1-4)MurNAc, the repeating structure from bacterial cell wall peptidoglycan, and the Man beta-D-(1-4)GlcNAc reducing-end component of glycoproteins. Km and Vm for hydrolysis of these substrates were of similar magnitude as for di-N-acetylchitobiose (6.3 mM and 15 mumol/min/mg protein, respectively). Liver tissues from nin mammalian species were surveyed for the presence of chitobiase activity. The activity was found in rat, mouse, rabbit, and guinea pig liver (Stirling [(1974) FEBS Lett. 39, 171-175] previously observed the enzyme in human liver), but not in dog, sheep, pig, cat, and cow liver. The presence or absence of chitobiase so far observed was found to exactly correlate with the type of oligosaccharide fragments found to accumulate in animals containing genetic or inhibitor-induced lysosomal storage pathologies. The presence of the chitobiase corresponds to the occurrence of one GlcNAc unit at the reducing end of stored oligosaccharides, while the absence of this glycosidase yields fragments with an intact GlcNAc beta-D-(1-4)GlcNAc moiety. These results verify our previous proposal that lysosomal disassembly of glycoproteins to free amino acids and sugars is an ordered, bidirectional pathway in which chitobiase (when present) catalyzes the last step during digestion of the protein-oligosaccharide linkage region.     

Purification of two glycoproteins expressing beta 1-6 branched Asn-linked oligosaccharides from metastatic tumour cells.

Increased branching at the trimannosyl core of 'complex-type' Asn-linked oligosaccharides has been observed in both human and murine tumour cells, and appears to be associated with enhanced metastatic potential in several murine tumour models [Dennis, Laferte, Waghorne, Breitman & Kerbel (1987), Science 236, 582-585]. The lectin leucoagglutinin (L-PHA) requires the-GlcNAc beta 1-6Man alpha 1-6Man-linked lactosamine antenna in complex-type oligosaccharides for high-affinity binding and can be used to detect these structures in glycoproteins separated on SDS/polyacrylamide-gel electrophoresis. The major L-PHA-binding glycoproteins in the highly metastatic lymphoid tumour cell line called MDAY-D2 were purified and resolved into two major species, termed P2A (110 kDa) and P2B (130 kDa). P2A had L-PHA-reactive Asn-linked oligosaccharides with polylactosamine sequences as well as a large component of sialylated O-linked carbohydrates. The glycoprotein showed structural characteristics similar to those of leukosialin (i.e. CD43), a glycoprotein previously identified on the surface of leukocytes. Based on monosaccharide compositional analysis and glycosidase digestions, P2B was found to be 50-60% Asn-linked oligosaccharide containing polylactosamine sequences and sialic acid. The N-terminal peptide sequence of P2B was determined to be very similar to that of murine lysosomal membrane glycoprotein (LAMP-1), a ubiquitous glycoprotein found largely in the lysosomal membranes but also in the plasma membrane of several murine and human tumour cell lines.     

The similar effects of swainsonine and locoweed on tissue glycosidases and oligosaccharides of the pig indicate that the alkaloid is the principal toxin responsible for the induction of locoism

A neurological condition resembling that observed in hereditary mannosidosis occurs in animals ingesting spotted locoweed and plants of the genus Swainsona. Swainsonine has been isolated from these plants and has been suggested to be the primary causative agent in inducing the pathological condition. This alkaloid has also been found to increase tissue acid alpha-D-mannosidase levels in rats while lowering liver Golgi mannosidase II levels. In the present study, the effects of locoweed and swainsonine were directly compared for the first time, with the pig as experimental animal. Both increased most lysosomal acid glycosidase activities in most tissues, decreased liver Golgi mannosidase II levels, increased plasma hydrolase levels, and greatly increased tissue oligosaccharide, especially Man5GlcNAc2 and Man4GlcNAc2. These results indicate that swainsonine is the agent in locoweed responsible for the enzymatic and oligosaccharide changes. The behavior of the animals was also similarly affected by swainsonine and locoweed.     

Swainsonine affects the processing of glycoproteins in vivo

Rats, sheep and guinea pigs treated with swainsonine excrete 'high mannose' oligosaccharides in urine. The major rat and guinea pig oligosaccharide is (Man)5GlcNAc, whereas sheep excrete a mixture of oligosaccharides of composition (Man)2-5GlcNAc2 and (Man)3-5GlcNAc. The presence of these oligosaccharides suggests that Golgi alpha-D-mannosidase II as well as lysosomal alpha-D-mannosidase is inhibited by swainsonine resulting in storage of abnormally processed asparagine-linked glycans from glycoproteins. Altered glycoprotein processing appears to have little effect on the health of the intoxicated animal, but the accompanying lysosomal storage produces a disease state.     

Carbohydrate mapping by mass spectrometry: a novel method for identifying attachment sites of Asn-linked sugars in glycoproteins

A new method is described for locating the specific sites of attachment of Asn-linked carbohydrates in glycoproteins. The molecular weights of peptides released from the glycoprotein with proteases of known specificity are determined by fast atom bombardment mass spectrometry and fitted to the known or DNA-derived sequence. Oligosaccharides attached to Asn are released either before or after proteolysis with a glycosidase, usually peptide: N-glycosidase F, an enzyme that cleaves the beta-aspartylglycosylamine linkage of all known types of Asn-linked sugars and converts the attachment-site Asn to Asp. New peaks appearing in the mass spectra after treatment with glycosidase correspond to formerly glycosylated sites. Conversely, signals which disappear after glycosidase treatment correspond to glycopeptides. The differences in mass between these sets of signals define the composition of the carbohydrate at the given site in terms of deoxyhexose, hexose, N-acetylhexosamine, and sialic acid content. The extent of glycosylation at a given site can be estimated from the ratio of the peak heights corresponding to the Asn- vs Asp-containing peptides which differ by 1 Da in mass. This rapid and sensitive (low nmol) technique is illustrated here for ribonuclease B and for tissue plasminogen activator, a multiply glycosylated glycoprotein.     

Aspartylglycosaminuria: biochemistry and molecular biology

Aspartylglucosaminuria (AGU, McKusick 208400) is an autosomal recessive lysosomal storage disease caused by defective degradation of Asn-linked glycoproteins. AGU mutations occur in the gene (AGA) for glycosylasparaginase, the enzyme necessary for hydrolysis of the protein oligosaccharide linkage in Asn-linked glycoprotein substrates undergoing metabolic turnover. Loss of glycosylasparaginase activity leads to accumulation of the linkage unit Asn-GlcNAc in tissue lysosomes. Storage of this fragment affects the pathophysiology of neuronal cells most severely. The patients notably suffer from decreased cognitive abilities, skeletal abnormalities and facial grotesqueness. The progress of the disease is slower than in many other lysosomal storage diseases. The patients appear normal during infancy and generally live from 25 to 45 years. A specific AGU mutation is concentrated in the Finnish population with over 200 patients. The carrier frequency in Finland has been estimated to be in the range of 2.5-3% of the population. So far there are 20 other rare family AGU alleles that have been characterized at the molecular level in the world's population. Recently, two knockout mouse models for AGU have been developed. In addition, the crystal structure of human leukocyte glycosylasparaginase has been determined and the protein has a unique alphabetabetaalpha sandwich fold shared by a newly recognized family of important enzymes called N-terminal nucleophile (Ntn) hydrolases. The nascent single-chain precursor of glycosylase araginase self-cleaves into its mature alpha- and beta-subunits, a reaction required to activate the enzyme. This interesting biochemical feature is also shared by most of the Ntn-hydrolase family of proteins. Many of the disease-causing mutations prevent proper folding and subsequent activation of the glycosylasparaginase.     

Inhibitory effect of pseudo-aminosugars on oligosaccharide glucosidases I and II and on lysosomal alpha-glucosidase from rat liver

We examined the inhibitory effect of three pseudo-aminosugars (validamine, valienamine, and valiolamine), which were isolated from the broth of Streptomyces hygroscopicus, on the oligosaccharide-processing glucosidases I and II involved in glycoprotein biosynthesis in rat liver. Both glucosidases I and II were inhibited to the same extent by the pseudoaminosugars, and valiolamine had a more potent inhibitory activity than validamine or valienamine. A 50% inhibition of valiolamine was observed at 12 microM for glucosidase I and glucosidase II activities acting respectively on the substrates Glc3Man9GlcNAc2 and p-nitrophenyl alpha-D-glucopyranoside. Further, in order to investigate further the ability of valiolamine to inhibit glucosidase I, reaction products were analyzed by gel filtration on a Bio-Gel P-4 column. We also compared the inhibitory action of these pseudo-aminosugars on the acid alpha-glucosidase of rat liver lysosomes. They competitively inhibited the hydrolysis of both substrates, maltose and glycogen. Valiolamine again had a more potent lysosomal alpha-glucosidase inhibitory activity than the other two. The Ki values of valiolamine for the hydrolysis of maltose and glycogen were 8.1 and 11 microM, respectively. Valiolamine is a particularly effective inhibitor of oligosaccharide glucosidases I and II and of lysosomal alpha-glucosidase. Hence valiolamine might be useful as a research tool in investigations of carbohydrate metabolism.     

Evidence for two catabolic endoglycosidase activities in β-mannosidase-deficient goat fibroblasts

Cultured skin fibroblasts derived from Nubian goats deficient in lysosomal β-mannosidase, which had previously been shown to accumulate storage oligosaccharides with the structures Manβ4GlcNAcβ4GlcNAc and Manβ4GlcNAc (in the ratio of 2.7:1) were evaluated for their ability to catabolize exogenous [³H]GlcN-labelled glycoproteins isolated from the secretions of cultured goat or human fibroblasts. Regardless of the source of exogenous labelled glycoprotein, affected goat fibroblasts took up the labelled glycoprotein from the culture medium and subsequently accumulated the same major labelled oligosaccharide, identified as Manβ4GlcNAcβ4GlcNAc; no such oligosaccharide accumulated in normal goat fibroblasts under the same conditions. Tunicamycin-treated affected fibroblasts also took up labelled exogenous glycoprotein and accumulated labelled storage trisaccharide, further suggesting the direct accumulation of storage trisaccharide from impaired glycoprotein-associated oligosaccharide catabolism. Treatment of metabolically labelled affected fibroblasts with leupeptin, an inhibitor of lysosomal cathepsins, resulted in the 2- to 6-fold inhibition of trisaccharide accumulation, while having little effect on the uptake of [³H]GlcN or the accumulation of labelled disaccharide. The results are most consistent with the presence of two endoglycosidases, an endo-β-N-acetylglucosaminidase and an endo-aspartylglucosaminidase, in goat fibroblasts. These two activities, rather than heterogeneous core oligosaccharide structures, are responsible for the ultimate accumulation of storage oligosaccharides with one and two GlcNAc residues at their reducing terminus.     

alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene inhibition of rat liver alpha-D-mannosidases

The compound alpha-D-mannopyranosylmethyl-p-nitrophenyltriazene (alpha-ManMNT) has been tested for its effect on four alpha-D-mannosidase activities present in rat liver. When p-nitrophenyl alpha-D-mannopyranoside was used as a substrate, preincubation of enzyme with 1.0 mM alpha-ManMNT inhibited soluble alpha-D-mannosidase by 90%, lysosomal alpha-D-mannosidase by approx. 60%, and had virtually no effect on Golgi mannosidase II. Golgi mannosidase I removal of the four alpha-1,2-linked D-mannoses from the common Man9GlcNAc2 oligosaccharide structure formed during N-linked glycoprotein biosynthesis was also blocked by treatment of the Golgi fraction with this compound. Mannosyltriazene inhibition of the three susceptible hepatic alpha-D-mannosidases was largely irreversible. alpha-ManMNT should therefore be useful for studying oligosaccharide processing and possibly for determining the turnover time of the inhibited alpha-D-mannosidases.     

UDP-GlcNAc:Glycoprotein GlcNAc-Transferase is Located in the Golgi Apparatus of Developing Bean Cotyledons

The transport and accumulation of phytohemagglutinin in developing bean (Phaseolus vulgaris L.) cotyledons is accompanied by the transient presence of N-acetylglucosamine (GlcNAc) residues on the oligosaccharide sidechains of this glycoprotein. These peripheral GlcNAc residues can be distinguished from those in the chitobiose portion of the oligosaccharide sidechains by their sensitivity to removal by the exoglycosidase β-N-acetylglucosaminidase. GlcNAc residues sensitive to removal by β-N-acetylglucosaminidase are present not only on phytohemagglutinin, but also on other newly synthesized proteins. The enzyme UDPGlcNAc:glycoprotein GlcNAc-transferase which transfers GlcNAc residues to glycoproteins was first described by Davies and Delmer (Plant Physiol 1981 68: 284-291). The data presented here show that this enzyme is associated with the Golgi complex of developing cotyledons.     

Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices

Incubations of thyroid microsomes with radiolabeled dolichyl pyrophosphoryl oligosaccharide (Glc3Man9-GlcNAc2) under conditions optimal for the N-glycosylation of protein resulted in the release, by apparently independent enzymatic reactions, of two types of neutral glucosylated polymannose oligosaccharides which differed from each other by terminating either in an N-acetylglucosamine residue (Glc3Man9GlcNAc1) or a di-N-acetylchitobiose moiety (Glc3Man9GlcNAc2). The first mentioned oligosaccharide, which was released in a steady and slow process unaffected by the addition of EDTA, appeared to be primarily the product of endo-beta-N-acetylglucosaminidase action on newly synthesized glycoprotein and such an enzyme with a neutral pH optimum capable of hydrolyzing exogenous glycopeptides and oligosaccharides (Km = 18 microM) was found in the thyroid microsomal fraction. The Glc3Man9GlcNAc2 oligosaccharide, in contrast, appeared to originate from the oligosaccharide-lipid by a rapid hydrolysis reaction which closely paralleled the N-glycosylation step, progressing as long as oligosaccharide transfer to protein occurred and terminating when carbohydrate attachment ceased either due to limitation of lipid-saccharide donor or addition of EDTA. There was a striking similarity between oligosaccharide release and transfer to protein with lipid-linked Glc3Man9GlcNAc2 serving as a 10-fold better substrate for both reactions than lipid-linked Man9-8GlcNAc2. The coincidence of transferase and hydrolase activities suggest the possibility of the existence of one enzyme with both functions. The physiological relevance of oligosaccharide release was indicated by the formation of such molecules in thyroid slices radiolabeled with [2-3H]mannose. Large oligosaccharides predominated (12 nmol/g) and consisted of two families of components; one group terminating in N-acetylglucosamine, ranged from Glc1Man9GlcNAc1 to Man5GlcNAc1 while the other contained the di-N-acetylchitobiose sequence and included Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, and Man9GlcNAc2.     

Structural studies on a binding site for Dolichos biflorus agglutinin in the small intestine of the mouse

Glycoproteins which bound to Dolichos biflorus agglutinin (DBA) were isolated from the small intestine of 129/Sv mice. Among oligosaccharides released from the carbohydrate moieties of the glycoproteins by endo-beta-galactosidase, the major one with N-acetylgalactosamine at the non-reducing end was isolated by QAE-Sephadex A-25 column chromatography. The structure of the oligosaccharide was elucidated to be GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc beta 1----3Gal by compositional analysis, methylation analysis before and after mild acid hydrolysis, sequential glycosidase digestion, secondary ion mass spectrometry (SIMS), and nuclear magnetic resonance spectroscopy. The SIMS signal of m/z 1,071 was consistent with the presence of the branched sequence, GalNAc(NeuAc)GalGlcNAc, and the signal was also detected in the high-molecular-weight fraction obtained after endo-beta-galactosidase digestion. The pentasaccharide identified here has the terminal structure of ganglioside GM2, and an apparently identical one has been identified as the epitope of blood group Sda and the DBA binding site in human T-H urinary glycoprotein. Thus, the present result has extended our knowledge of the biological meaning of the oligosaccharide structure and has established that GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc is a DBA binding site in the small intestine of the mouse.     

Inhibitors of the biosynthesis and processing of N-linked oligosaccharides

A number of glycoproteins have oligosaccharides linked to protein in a GlcNAc----asparagine bond. These oligosaccharides may be either of the complex, the high-mannose or the hybrid structure. Each type of oligosaccharides is initially biosynthesized via lipid-linked oligosaccharides to form a Glc3Man9GlcNAc2-pyrophosphoryl-dolichol and transfer of this oligosaccharide to protein. The oligosaccharide portion is then processed, first of all by removal of all three glucose residues to give a Man9GlcNAc2-protein. This structure may be the immediate precursor to the high-mannose structure or it may be further processed by the removal of a number of mannose residues. Initially four alpha 1,2-linked mannoses are removed to give a Man5 - GlcNAc2 -protein which is then lengthened by the addition of a GlcNAc residue. This new structure, the GlcNAc- Man5 - GlcNAc2 -protein, is the substrate for mannosidase II which removes the alpha 1,3- and alpha 1,6-linked mannoses . Then the other sugars, GlcNAc, galactose, and sialic acid, are added sequentially to give the complex types of glycoproteins. A number of inhibitors have been identified that interfere with glycoprotein biosynthesis, processing, or transport. Some of these inhibitors have been valuable tools to study the reaction pathways while others have been extremely useful for examining the role of carbohydrate in glycoprotein function. For example, tunicamycin and its analogs prevent protein glycosylation by inhibiting the first step in the lipid-linked pathway, i.e., the formation of Glc NAc-pyrophosphoryl-dolichol. These antibiotics have been widely used in a number of functional studies. Another antibiotic that inhibits the lipid-linked saccharide pathway is amphomycin, which blocks the formation of dolichyl-phosphoryl-mannose. In vitro, this antibiotic gives rise to a Man5GlcNAc2 -pyrophosphoryl-dolichol from GDP-[14C]mannose, indicating that the first five mannose residues come directly from GDP-mannose rather than from dolichyl-phosphoryl-mannose. Other antibodies that have been shown to act at the lipid-level are diumycin , tsushimycin , tridecaptin, and flavomycin. In addition to these types of compounds, a number of sugar analogs such as 2-deoxyglucose, fluoroglucose , glucosamine, etc. have been utilized in some interesting experiments. Several compounds have been shown to inhibit glycoprotein processing. One of these, the alkaloid swainsonine , inhibits mannosidase II that removes alpha-1,3 and alpha-1,6 mannose residues from the GlcNAc- Man5GlcNAc2 -peptide. Thus, in cultured cells or in enveloped viruses, swainsonine causes the formation of a hybrid structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

A di-N-acetylchitobiase activity is involved in the lysosomal catabolism of asparagine-linked glycoproteins in rat liver

A perfused rat liver was used to study the effects of 5-diazo-4-oxo-L-norvaline on lysosomal glycoprotein catabolism. Addition of this compound (1.0 mM) to the perfusate reduced activity of beta-aspartyl-N-acetylglucosylamine amidohydrolase by 99% in 1 h. Treated livers were unable to completely degrade endocytosed N-acetyl[14C]glucosamine-labeled asialo-alpha 1-acid glycoprotein as evidenced by a 50% reduction in radiolabeled serum glycoprotein secretion compared to controls. This decreased degradation was matched by a lysosomal accumulation of glycopeptides with the structure: GlcNAc beta(1-4)GlcNAc-Asn. The result suggested the presence of an unrecognized glycosidase in rat liver lysosomes, since this remnant was extended by one more GlcNAc residue than would have been expected after specific inactivation of the amidohydrolase. Such a novel enzyme would therefore catalyze cleavage of the N-acetylglucosamine residue at the reducing end of alpha 1-acid glycoprotein oligosaccharides only following removal of the linking Asn. The activity was then detected in lysosomal extracts by using intact asialo-biantennary oligosaccharides labeled with [3H] galactose or N-acetyl[14C]glucosamine residues as a substrate. To prevent simultaneous digestion of the material from its nonreducing end, beta-D-galactosidase in the enzyme extract was first inactivated with the irreversible active site-directed inhibitor, beta-D-galactopyranosylmethyl-p-nitrophenyltriazene. The observed di-N-acetylchitobiose cleaving activity worked optimally at pH 3.4 and was uniquely associated with the lysosomal fraction of the liver homogenate. The enzyme also cleaved triantennary chains and di-N-acetylchitobiose, but failed to hydrolyze substrates that had been reduced with NaBH4. The new glycosidase was well separated from N-acetyl-beta-D-glucosaminidase (assayed with p-nitrophenyl-beta-D-glucosaminide) by gel filtration chromatography and had an apparent molecular weight of 37,000. A similar enzyme that hydrolyzes di-N-acetylchitobiose had previously been found in extracts of human liver (Stirling, J. L. (1974) FEBS Lett. 39, 171-175).     

Relative susceptibilities of the glucosamine-glucuronic acid and N-acetylglucosamine-glucuronic acid linkages to heparin lyase III

Heparin lyases are valuable tools for generating oligosaccharide fragments and in sequence determination of heparan sulfate (HS). Heparin lyase III is known to cleave the linkages between N-acetylglucosamine (GlcNAc) or N-sulfated glucosamine (GlcNS) and glucuronic acid (GlcA) as the primary sites and the linkages between GlcNAc, GlcNAc(6S), or GlcNS and iduronic acid as secondary sites. N-Unsubstituted glucosamine (GlcN) occurs as a minor component in HS, and it has been associated with various bioactivities. Here we investigate the specificity of heparin lyase III toward the GlcN-GlcA linkage using a recombinant enzyme of high purity and as substrates the partially de-N-acetylated polysaccharide of Escherichia coli K5 strain and derived hexasaccharides. The specificity of lyase III toward the GlcN-GlcA linkage is deduced by sequencing of the oligosaccharide products using electrospray mass spectrometry with collision-induced dissociation and MS/MS scanning. The results demonstrate that under controlled conditions for partial digestion, lyase III does not act at the GlcN-GlcA linkage, whereas GlcNAc-GlcA is cleaved. Even under forced conditions for exhaustive digestion, the GlcN-GlcA linkage is only partly cleaved. It is this property of lyase III that has enabled the isolation of a unique, nonsulfated antigenic determinant DeltaUA-GlcN-UA-GlcNAc from HS and from partially de-N-acetylated K5 polysaccharide. It was unexpected that pentasaccharide fragments were also detected among the digestion products of the K5 polysaccharide used. It is possible that these are products of an additional glycosidase activity of lyase III, although other mechanisms cannot be completely ruled out.     

Posttranslational Protein Modification: Biosynthetic Control Mechanisms in the Glycosylation of the Major Myelin Glycoprotein by Schwann Cells

The posttranslational processing of the asparagine-linked oligosaccharide chain of the major myelin glycoprotein (P0) by Schwann cells was evaluated in the permanently transected, adult rat sciatic nerve, where there is no myelin assembly, and in the crush injured nerve, where there is myelin assembly. Pronase digestion of acrylamide gel slices containing the in vitro labeled [3H]mannose and [3H]fucose P0 after electrophoresis permitted analysis of the glycopeptides by lectin affinity and gel filtration chromatography. The concanavalin A-Separose profile of the [3H]mannose P0 glycopeptides from the transected nerve revealed the high-mannose-type oligosaccharide as the predominant species (72.9%), whereas the normally expressed P0 glycoprotein that is assembled into the myelin membrane in the crushed nerve contains 82.9-91.9% of the [3H]mannose radioactivity as the complex-type oligosaccharide chain. Electrophoretic analysis of immune precipitates verified the [3H]mannose as being incorporated into P0 for both the transected and crushed nerve. The high-mannose-type glycopeptides of the transected nerve isolated from the concanavalin A-Sepharose column were hydrolyzed by endo-beta-N-acetylglucosaminidase H, and the oligosaccharides were separated on Biogel P4. Man8GlcNAc and Man7GlcNAc were the predominant species with radioactivity ratios of 12.5/7.2/1.4/1.0 for the Man8, Man7, Man6, and Man5 oligosaccharides, respectively. Jack bean alpha-D-mannosidase gave the expected yields of free Man and ManGlcNAc from these high-mannose-type oligosaccharides. The data support the notion that at least two alpha-1,2-mannosidases are responsible for converting Man9GlcNAc2 to Man5GlcNAc2. The present experiments suggest distinct roles for each mannosidase and that the second mannosidase (I-B) may be an important rate-limiting step in the processing of this glycoprotein with the resulting accumulation of Man8GlcNAc2 and Man7GlcNAc2 intermediates. Pulse chase experiments, however, demonstrated further processing of this high-mannose-type oligosaccharide in the transected nerve. The [3H]mannose P0 glycoprotein with Mr of 27,700 having the predominant high-mannose-type oligosaccharide shifted its Mr to 28,500 with subsequent chase. This band at 28,500 was shown to have the complex-type oligosaccharide chain and to contain fucose attached to the core asparagine-linked GlcNAc residue. The extent of oligosaccharide processing of this down-regulated glycoprotein remains to be determined.     

Cytosolic glycosidases: do they exist?

The substrate specificity of the alpha-D-mannosidases of rat liver lysosome and cytosol was examined using oligosaccharides of the oligomannosidic type. The hydrolysis products were characterized by 400 MHz 1H-NMR spectroscopy. Both catabolic pathways occur in ordered ways, but are quite different. In fact, the lysosomal pathway is a two-step process: the first step involves a Zn(2+)-independent alpha-1,2-mannosidase activity, whereas the second involves a Zn(2+)-dependent alpha-1,3- and alpha-1,6-mannosidase activity. The final product is the disaccharide Man(beta 1-4)GlcNAc. In contrast, the cytosolic pathway leads, in one step, to a unique hexasaccharide (Man5GlcNAc) which has the same structure as the polyprenolic intermediate synthesized on the cytosolic face of the rough endoplasmic reticulum during the biosynthesis of N-glycosylprotein glycans: Man(alpha 1-2)-Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)] Man(beta 1-4)GlcNAc(beta 1-4)-GlcNAc(alpha)P-P-Dol. In addition, the enzymatic parameters of lysosome, endoplasmic reticulum and cytosol alpha-D-mannosidases are quite different. These results lead to the conclusion that the cytosol contains specific alpha-D-mannosidases which do not originate from lysosomes nor from endoplasmic reticulum. The discovery of cytosolic endo-N-acetyl-beta-D-glucosaminidase active on 'immature complex glycans' (glycopeptides of the oligomannosidic type and of the desialylated N-acetyllactosaminic type) as well as on the glycosyl-dolichol pyrophosphate intermediates allows us to hypothesize that these enzymes belong to a control system of N-glycosylprotein biosynthesis, their role being to destroy unfinished glycans. The fate of the formed oligosaccharide structures is discussed: are they destroyed by cytosolic or lysosomal exoglycosidases, or do they carry an 'oligosaccharin-like activity'?     

Control of glycoprotein synthesis. UDP-GlcNAc:glycopeptide beta 4-N-acetylglucosaminyltransferase III, an enzyme in hen oviduct which adds GlcNAc in beta 1-4 linkage to the beta-linked mannose of the trimannosyl core of N-glycosyl oligosaccharides

Hen oviduct membranes are shown to catalyze the following enzyme reaction: GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-Asn + UDP-GlcNAc leads to GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)GlcNAc beta 1-4)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-Asn + UDP. The enzyme catalyzing this reaction has been named UDP-GlcNAc:glycopeptide beta 4-N-acetylglucosaminyltransferase III (GlcNAc-transferase III) to distinguish it from two other GlcNAc-transferases (I and II) present in hen oviduct and previously described in several mammalian tissues. GlcNAc-transferases I and II, respectively, attach GlcNAc in beta 1-2 linkage to the Man alpha 1-3 and Man alpha 1-6 arms of Asn-linked oligosaccharide cores. A specific assay for GlcNAc-transferase III was devised by using concanavalin A/Sepharose columns to separate the product of transferase III from other interfering radioactive glycopeptides formed in the reaction. The specific activity of GlcNAc-transferase III in hen oviduct membranes is about 5 nmol/mg of protein/h. Substrate specificity studies have shown that GlcNAc-transferase III requires both terminal beta 1-2-linked GlcNAc residues in its substrate for maximal activity. Removal of the GlcNAc residue on the Man alpha 1-6 arm reduces activity by at least 85% and removal of both GlcNAc residues reduces activity by at least 93%. Two large scale preparations of product were subjected to high resolution proton NMR spectroscopy to establish the incorporation by the enzyme of a GlcNAc in beta 1-4 linkage to the beta-linked Man. This GlcNAc residue is called a "bisecting" GlcNAc and appears to play important control functions in the synthesis of complex N-glycosyl oligosaccharides. Several enzymes in the biosynthetic scheme are unable to act on glycopeptide substrates containing a bisecting GlcNAc residue.     

Chemoenzymatic synthesis of N-linked neoglycoproteins through a chitinase-catalyzed transglycosylation

A novel application of the Bacillus sp. chitinase for the chemoenzymatic synthesis of N-linked neoglycoproteins is described. Three chitinases with different molecular size were purified from the crude chitinase preparation. The purified chitinases were evaluated for their hydrolytic and transglycosylation activity. One chitinase with a molecular size of 100 kDa (Chi100) was identified to be the one with highest transglycosylation/hydrolysis ratio. Chi100 could effectively recognize LacNAc-oxazoline and Manalpha1,3Glcbeta1,4GlcNAc-oxazoline as the donor substrate to glycosylate Asn-linked GlcNAc, while it was unable to recognize Manbeta1,4GlcNAc and Man(3)GlcNAc-oxazolines as the donor substrates. The chitinase-catalyzed transglycosylation was successfully extended to the remodeling of ribonuclease B to afford neoglycoproteins. Although the yield needs to be optimized, the chitinase-catalyzed transglycosylation provides a potentially useful tool for the synthesis of neoglycoproteins carrying novel N-linked oligosaccharides.     

Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing.

The biosynthesis of the various types of N-linked oligosaccharide structures involves two series of reactions: 1) the formation of the lipid-linked saccharide precursor, Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol, by the stepwise addition of GlcNAc, mannose and glucose to dolichyl-P, and 2) the removal of glucose and mannose by membrane-bound glycosidases and the addition of GlcNAc, galactose, sialic acid, and fucose by Golgi-localized glycosyltransferases to produce different complex oligosaccharide structures. For most glycoproteins, the precise role of the carbohydrate is still not known, but specific N-linked oligosaccharide structures are key players in targeting of lysosomal hydrolases to the lysosomes, in the clearance of asialoglycoproteins from the serum, and in some cases of cell:cell adhesion. Furthermore, many glycoproteins have more than one N-linked oligosaccharide, and these oligosaccharides on the same protein frequently have different structures. Thus, one oligosaccharide may be of the high-mannose type whereas another may be a complex chain. One approach to determining the role of specific structures in glycoprotein function is to use inhibitors that block the modification reactions at different steps, causing the cell to produce glycoproteins with altered carbohydrate structures. The function of these glycoproteins can then be assessed. A number of alkaloid-like compounds have been identified that are specific inhibitors of the glucosidases and mannosidases involved in glycoprotein processing. These compounds cause the formation of glycoproteins with glucose-containing high mannose structures, or various high-mannose or hybrid chains, depending on the site of inhibition. These inhibitors have also been useful for studying the processing pathway and for comparing processing enzymes from different organisms.