Separation of dolichylpyrophosphoryloligosaccharides by liquid chromatography

Fourteen dolichylpyrophosphoryloligosaccharides, precursors of the asparagine-linked oligosaccharides of glycoproteins, have been separated by liquid chromatography on silica gel. The dolichylpyrophosphoryl-N-acetylglucosamine and the dolichylpyrophosphoryl-(N-acetylglucosamine)₂-(mannose)₉(glucose)_2,3 thus resolved were shown to retain their activity as substrates in enzyme catalyzed reactions. The chromatography procedure for the first time makes available many of these single intermediates for further study.     

The mechanism of synthesis of the polysaccharide part of mannan in Saccharomyces cerevisiae

Saccharomyces cerevisiae wild-type and mutant cells affected in the structure of mannan outer chain were shown to possess in vivo one major dolichol-P-P-bound oligosaccharide. The size, monosaccharide composition, and pattern obtained upon acetolysis and paper chromatography of the oligosaccharide were the same for all strains and for the main corresponding compound isolated from animal tissues. Evidence is presented indicating that the dolichol-P-P derivative occurring in vivo, and containing 2-N-acetylglucosamine, 9-mannose, and 3-glucose residues, is the intermediate involved in yeast protein glycosylation. The transfer of the oligosaccharide to protein was followed in vivo by the excision of the glucose and at the most one mannose residue. Mannoses were then added to the trimmed saccharide moiety. No difference between the first stages (i.e., excision of monosaccharides) of the processing of the protein-bound oligosaccharides by wild-type and mutant cells was found. However, mutants carrying the mnn 1 mutation, which are known to be devoid of terminal α(1–3)-linked mannose residues in the mannan outer chain and inner core, were found not to add such mannose residues to the already glucose-free protein-bound oligosaccharide.     

Abnormal processing of the modified oligosaccharide side chains of phytohemagglutinin in the presence of swainsonine and deoxynojirimycin

Phytohemagglutinin, the glycoprotein lectin of the common bean, Phaseolus vulgaris, has both high-mannose (Man_8-9GlcNAc₂) and modified oligosaccharide side chains. The modified side chains have glucosamine, mannose, fucose, and xylose in the molar ratios 2:3.8:0.6:0.5, and are resistant to hydrolysis by endoglycosidase H. Synthesis and processing of side chains in the presence of 1-deoxynojirimycin, an inhibitor of α-glucosidase, results in the formation of chains which are all alike. They are sensitive to endoglycosidase H, do not contain fucose, and are largely resistant to α-mannosidase. This indicates that they are probably high-mannose chains blocked by terminal glucose residues. Synthesis and processing of side chains in the presence of swainsonine, an inhibitor of α-mannosidase II, results in the formation of normal high-mannose chains, and of modified chains which contain fucose residues, are resistant to endoglycosidase H, and can be distinguished from normal modified chains only by the presence of extra mannose residues.

Processing of the phytohemagglutinin modified chains of PHA under normal conditions involves the attachment of peripheral N-acetylglucosamine residues in the Golgi complex and their subsequent removal in the protein bodies. The attachment of the N-acetylglucosamine residues is largely inhibited by deoxynojirimycin but still occurs in the presence of swainsonine. The results presented in this work show that processing of the asparagine-linked oligosaccharides is under the control of several glycosidases and glycosyltransferases and involves the formation of intermediate products.

     

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

Structural insights into the substrate specificity of Streptococcus pneumoniae β(1,3)-galactosidase BgaC

The surface-exposed β-galactosidase BgaC from Streptococcus pneumoniae was reported to be a virulence factor because of its specific hydrolysis activity toward the β(1,3)-linked galactose and N-acetylglucosamine (Galβ(1,3)NAG) moiety of oligosaccharides on the host molecules. Here we report the crystal structure of BgaC at 1.8 Å and its complex with galactose at 1.95 Å. At pH 5.5–8.0, BgaC exists as a stable homodimer, each subunit of which consists of three distinct domains: a catalytic domain of a classic (β/α)₈ TIM barrel, followed by two all-β domains (ABDs) of unknown function. The side walls of the TIM β-barrel and a loop extended from the first ABD constitute the active site. Superposition of the galactose-complexed structure to the apo-form revealed significant conformational changes of residues Trp-243 and Tyr-455. Simulation of a putative substrate entrance tunnel and modeling of a complex structure with Galβ(1,3)NAG enabled us to assign three key residues to the specific catalysis. Site-directed mutagenesis in combination with activity assays further proved that residues Trp-240 and Tyr-455 contribute to stabilizing the N-acetylglucosamine moiety, whereas Trp-243 is critical for fixing the galactose ring. Moreover, we propose that BgaC and other galactosidases in the GH-35 family share a common domain organization and a conserved substrate-determinant aromatic residue protruding from the second domain.     

Subcellular localization of glycosidases and glycosyltransferases involved in the processing of N-linked oligosaccharides

Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc₃Man₉(GlcNAc)₂, was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associated with the Golgi. These include mannosidase I (removes 1-2 mannose residues from Man_6-9[GlcNAc]₂), mannosidase II (removes mannose residues from GlcNAcMan₅[GlcNAc]₂), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). We have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltransferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities.     

Incorporation of glucose into lipid-linked saccharides in aorta and its inhibition by amphomycin

The particulate enzyme from pig aorta catalyzed the transfer of glucose from UDP-glucose into glucosyl-phosphoryl-dolichol, into lipid-linked oligosaccharides, and into glycoprotein. Radioactive lipid-linked oligosaccharides were prepared by incubating the extracts with GDP-[¹⁴C]mannose and UDP-[³H]glucose. When the labeled oligosaccharides were run on Bio-Gel P-4, the two different labels did not exactly coincide; the ³H peak eluted slightly earlier indicating that it was of higher molecular weight than the ¹⁴C material, but there was considerable overlap. The purified oligosaccharide(s) contained glucose, mannose, and N-acetylglucosamine but the ratios of these sugars varied from one enzyme preparation to another, probably depending on the endogenous oligosaccaride-lipids present in the microsomal preparation. Treatment of the [³H]glucose-labeled oligosaccharide with α-mannosidase gave rise to a ³H-labeled oligosaccharide which moved somewhat faster on Bio-Gel P-4 than the original oligosaccharide, suggesting it had lost one or two sugar residues. These data indicate that mannose and glucose are in the same oligosaccharide. The antibiotic, amphomycin, inhibited the transfer of glucose from UDP-glucose into the lipid-linked saccharides. However the synthesis of glucosyl-phosphoryl-dolichol was much more sensitive then was the synthesis of lipid-linked oligosaccharides. The glucose-labeled oligosaccharide produced in the absence of amphomycin was of high molecular weight based on paper chromatography. But in the presence of partially inhibitory concentrations of antibiotic, the oligosaccharide migrated more rapidly on paper chromatograms. However, amphomycin had no effect on the synthesis of glucosyl-ceramide by the aorta extracts. In fact, the antibiotic may stimulate glucosyl-ceramide by making more of the substrate, UDP-glucose, available for synthesis of this lipid.     

Glycoprotein biosynthesis in plants. Formation of lipid-linked oligosaccharides of mannose and N-acetylglucosamine by mung bean seedlings.

Cell-free enzyme particles from mung bean seedlings catalyze the incorporation of mannose from GDP-[¹⁴C]mannose and GlcNAc from UDP-[³H]GlcNAc into glycolipids and into glycoprotein. The most rapidly labeled product from GDP-mannose was characterized as a mannosyl-phosphoryl-polyisoprenol, whereas that from UDP-GlcNAc was a mixture of GlcNAc-(pyro)phosphoryl-polyisoprenol and a disaccharide composed of two N-acetylglucosamine residues attached to the polyisoprenol by a phosphoryl or pyrophosphoryl linkage. Radioactivity from GDP-mannose and UDP-GlcNAc was also incorporated into more polar lipids which have been partially characterized as a series of oligosaccharide-(pyro)phosphoryl-lipids. The mannose-labeled oligosaccharides released from these lipids by mild acid hydrolysis were found to contain GlcNAc at their reducing end indicating that these oligosaccharides contain both GlcNAc and mannose. Both the GlcNAc-labeled and the mannose-labeled oligosaccharides gave multiple radioactive peaks upon paper chromatography indicating that they are composed of a series of different sized oligosaccharides. Finally, radioactivity from GDP-[¹⁴C]mannose and UDP-[³H]GlcNAc is incorporated into an insoluble component. Ten percent of the mannose label and all of the GlcNAc label in this insoluble material could be solubilized by digestion with Pronase. The glycopeptides released by Pronase digestion appeared to be approximately the same size as the oligosaccharides from the lipid-linked oligosaccharides based on gel filtration chromatography on Sephadex G-50. The results are consistent with a mechanism for glycoprotein synthesis involving lipid-linked oligosaccharide intermediates.     

Peptide-N 4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducingN-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man₃(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and¹H- and¹³C-NMR assignments for the oligosaccharide Man₃(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).Abbreviations Endo endo--N-acetylglucosaminidase - Fuc fucose - GlcNAc N-acetylglucosamine - Man mannose - NMR nuclear magnetic resonance - PNGase peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase - Xyl xylose     

Glycosylation of endogenous lipids and proteins by preparations of chicken embryo fibroblasts

Glycosylation of endogenous phosphoisoprenyl lipids and membrane-associated proteins was shown to occur in preparations of chicken embryo fibroblasts incubated with GDP[¹⁴C]mannose and UDP-N-acetylglucosamine. The two preparations used were cells released from the culture dishes by buffered saline containing EDTA and crude membranes from those cells. Both β-mannosyl-phosphoryldolichol and oligosaccharide-phosphoryl lipids with five to eight sugar residues were labelled under the conditions employed. The oligosaccharide isolated from the octasaccharide-lipid fraction was shown to be heterogeneous after an analysis of the products formed by treatment of the oligosaccharide with glycosidases. Some of the oligosaccharides appeared to contain N-acetylglucosamine at positions external to that of [¹⁴C]mannose. Lipids with oligosaccharide moieties of different structures were made by the two preparations. The results of pulse-chase experiments were consistent with the glycosylated lipids being intermediates in glycoprotein biosynthesis.     

The periodate oxidation of bovine bone sialoprotein, and some observations on its structure

1. Bovine bone sialoprotein (mol.wt. 23000) contains N-acetylneuraminic acid and N-glycollylneuraminic acid, fucose, galactose, mannose, N-acetylgalactosamine and N-acetylglucosamine residues in the form of a very small number, perhaps one, of highly branched oligosaccharide structures linked covalently to peptide. 2. Periodate oxidation of the sialoprotein results in quantitative destruction only of the sialic acid and fucose residue consistent with the earlier findings of their positions as terminal groups. 3. Terminal sialic acid residues are attached to galactopyranose residues by 2,3-linkages, and to some N-acetylgalactosamine residues (at C-6). 4. Sequential Smith degradation indicates that N-acetylgalactosamine residues may be present as points of branching (linked in C-1, C-3 and C-6) and N-acetylglucosamine residues are located in the inner part of the structure, adjacent to the carbohydrate–peptide bond(s). 5. Mannose residues appear to be linked in the 1,3-positions.     

Activation of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae by various sugars

Endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae was activated by the addition of glucose, mannose, N-acetylglucosamine, and β-allose. While the enzyme did not appear to be significantly affected by the addition of galactose or N-acetylgalactosamine. These results indicate that the C-4 and C-6 positions of the monosaccharide are the most important for enzyme activation. Moreover, the enzyme was activated by the addition of disaccharides such as cellobiose, gentiobiose, and di-N-acetylchitobiose, but not by polysaccharides such as starch and yeast mannan. In the presence of N-acetylglucosamine, the enzyme activation occurred well over pH 4.0 and the K_m value of the enzyme for (Man)₆(GlcNAc)₂-Asn-dansyl changes from 1.2 mM to 3.2 mM.     

Evidence for multiple enzymes in the dolichol utilizing pathway of glycoprotein biosynthesis

A comparison has been made of the enzymes catalyzing the transfer of manose, glucose and N-acetylglucosamine from, respectively, GDPmannose, UDP-glucose and UDP-N-acetylglucosamine to endogenous dolichol phosphate (Dol-P) in liver Golgi membranes. Evidence is presented which suggests that all three reactions utilize the same pool of Dol-P. The transfer of mannose from GDP-Man to Dol-P is not inhibited by 0.1 mM UDP or UMP; 0.1 mM GDP did block the accumulation of mannose in Dol-P-Man. The net transfer of glucose and N-acetylglucosamine to Dol-P is prevented by 0.1 mM UDP but not 0.1 mM GDP. UDPglucose inhibits the reverse of the glucose transfer reaction but not reverse of the N-acetylglucosamine or mannose transfer reaction. On the basis of this, and other data, it is concluded that the three sugar transfer reactions utilize separate enzymes.     

Biosynthesis of glycoproteins in human placenta: Processing of oligosaccharides

The processing of the high-mannose asparagine-linked oligosaccharides synthesized by first-trimester human placenta has been investigated. Tissue was pulsed for 1 h with [2-³H]mannose and chased for zero, 45, 90, and 180 min in media containing unlabeled mannose. Glycopeptides, prepared by Pronase digestion of the delipidated membrane pellets at each time point, were treated with endo-β-N-acetylglucosaminidase-H to release the high-mannose asparagine-linked oligosaccharides. The largest major processing intermediate isolated was Glc₁Man₉GlcNAc, which was converted into Man₉GlcNAc, and then into Man₈GlcNAc, Man₇GlcNAc, Man₆GlcNAc, and Man₅GlcNAc. There was also a minor pathway in which mannosyl residues were removed prior to the glucose. By carrying out the detailed structural characterization of the individual processing intermediates, it was possible to demonstrate that processing of the Man₉GlcNAc to Man₅GlcNAc proceeded by the nonrandom removal of the α1,2-linked mannosyl residues. Specifically, of 12 possible sequences of removal of the four α1,2-linked mannosyl residues present in Man₉GlcNAc, first-trimester human placenta utilized only two of these in the processing of asparagine-linked oligosaccharides. It is suggested that the limited number of processing pathways reflects a high degree of specificity of these reactions in human placenta.     

Carbohydrate compositions of the rabbit plasminogen isozymes.

The carbohydrate compositions of the two affinity-chromatography-resolved isozymes of rabbit plasminogen and plasmin as well as the isoelectric-focusing-resolved subforms of each plasminogen isozyme have been investigated in detail. The first plasminogen isozyme as well as its subforms all possess four to five residues of N-acetylglucosamine, two residues of N-acetylgalactosamine, three residues of mannose and five residues of galactose per molecule of protein. Additionally, we previously reported three residues of sialic acid present on this protein molecule. The corresponding plasmin heavy chain for this isozyme contains essentially all of the carbohydrate, and the plasmin light chain appears devoid of carbohydrate. On the other hand, the second plasminogen isozyme as well as its subforms all possess only trace amounts of N-acetylglucosamine, two residues of N-acetylgalactosamine, less than one residue of mannose and three residues of galactose per molecule of protein. In addition, we have previously reported two residues of sialic acid for this molecule. Here, also, all carbohydrate appears on the heavy chain of the plasmin, which is prepared by activation of this particular plasminogen. Thus, the carbohydrate differences which we reported earlier in rabbit plasminogen isozymes are confirmed and extended.     

Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with α-methylmannoside, constitute about 25–30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chrolide columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with α-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.     

Monosaccharide Sequence of Protein-Bound Glycans of Uukuniemi Virus

Uukuniemi virus, a member of the Bunyaviridae family, was grown in BHK-21 cells in the presence of [³H]mannose. The purified virions were disrupted with sodium dodecyl sulfate and digested with pronase. The [³H]mannose-labeled glycopeptides of the mixture of the two envelope glycoproteins G1 and G2 were characterized by degrading the glycans with specific exo-and endoglycosidases, by chemical methods, and by analyzing the products with lectin affinity and gel chromatography. The glycopeptides of Uukuniemi virus fell into three categories: complex, high-mannose type, and intermediate. The complex glycopeptides probably contained mainly two NeuNAc-Gal-GlcNAc branches attached to a core (Man)₃(GlcNAc)₂ peptide. The high-mannose-type glycans were estimated to contain at least five mannose units attached to two N-acetylglucosamine residues. Both glycan species appeared to be similar to the asparagine-linked oligosaccharides found in many soluble and membrane glycoproteins. The results suggested that the intermediate glycopeptides contained a mannosyl core. In about half of the molecules, one branch appeared to be terminated in mannose, and one appeared to be terminated in N-acetylglucosamine. Such glycans are a novel finding in viral membrane proteins. They may represent intermediate species in the biosynthetic pathway from high-mannose-type to complex glycans. Their accumulation could be connected with the site of maturation of the members of the Bunyaviridae family. Electron microscopic data suggest that the virions bud into smooth-surfaced cisternae in the Golgi region. The relative amounts of [³H]mannose in the complex, high-mannose-type, and intermediate glycans were 25, 62, and 13%, respectively, which corresponded to the approximate relative number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly equimolar mixture of G1 and G2. Endoglycosidase H digestion of isolated [³⁵S]methionine-labeled G1 and G2 proteins suggested that most of the complex and intermediate chains were attached to G1 and that most of the high-mannose-type chains were attached to G2.     

Determination of the structure of the carbohydrate chains of acid α-glucosidase from human placenta

Acid α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) from human placenta (70 and 76 kDa) was found to contain 4 N-glycosidic carbohydrate chains per molecule. Sugar analysis of purified enzyme revealed the presence of mannose, N-acetylglucosamine and fucose at a molar ratio of 5.0:2.0:0.6. In addition, trace amounts of galactose and N-acetylneuraminic acid were detected. The sugar chains were liberated from the polypeptides by the hydrazinolysis procedure and subsequently fractionated by gel filtration and HPLC. Purified compounds were investigated by 500-MHz ¹H-NMR spectroscopy. Oligomannoside-type chains of intermediate size, e.g., Man₅GlcNAcGlcNAc-ol and Man₇GlcNAcGlcNAc-ol, and N-type chains of smaller size e.g., Man_2–3GlcNAc[Fuc]_0–1GlcNAc-ol, were demonstrated to be present at a ratio of 2:3. In addition, a small amount of sialylated N-acetyllactosamine-type chains has been found. The possible biosynthetic route of the fucose-containing small-size chains is discussed.     

Observations on the carbohydrate moiety of bovine pancreatic deoxyribonuclease A

The carbohydrate side chain of bovine pancreatic deoxyribonuclease A, which is attached to asparagine residue 18, contains two residues of N-acetylglucosamine proximal to the peptide chain followed by a variable number of mannose residues (4–10). The oligosaccharide structure bears a similarity to that in bovine pancreatic ribonuclease B. The present sequence studies have made use of α-mannosidase chromatographically purified from jack bean meal.     

Endo β-N-acetylglucosaminidase F cleavage specificity with peptide free oligosaccharides

Endo β-N-acetylglucosaminidase activities were determined based on conversion of oligosaccharides containing two N-acetylglucosamines to the oligosaccharides with a single N-acetylglucosamine at the reducing terminal and following their separation on a carbohydrate analyzer. The oligosaccharides eluted from the high performance anion exchange column in the order of fucosyl-N,N′ -diacetylchitobiose, N,N′ -diacetylchitobiose and N-acetylglucosamine containing reducing terminals. Using this assay, differences in cleavage specificity of the glycoproteins was determined. The commercial Endo F-peptide N-glycosidase/glycanyl amidase (PNGase)mixture readily leaved high mannose and complex oligosaccharides (neutral and sialyated) with common core α1–6 linked fucose found in porcine thyroglobulin including the trimannosyl-chitobiose core structure. However, the same Endo F mixture did not cleave the non-fucosylated complex oligosaccharides found in human transferrin and also the common core structure. Glycopeptide counterparts with and without fucose were good substrates for the endoglycosidases. These results show that the specificity of these enzymes is such that they can recognize the conformational differences between free oligosaccharides and glycopeptides with and without the common core α1–6 linked fucose. In contrast, highly purified Endo F cleaved only the high mannose type oligosaccharides and was unable to cleave ovalbumin hybrid type oligosaccharides. However, it was similar to Endo H when reduced ovalbumin oligosaccharides were used as substrates, consistent with the recently isolated Endo F subfraction F₁ being similar to Endo H [Trimble, R. B. and Tarentino, a. L. (1991). J. Biol. Chem. 266, 1646]. Results obtained in this study suggest that the complex oligosaccharides cleaving enzymes F₂ and F₃ show high specificity towards peptide free oligosaccharides with the core α1-6 linked fucose, unlike the glycopeptide substrates. Therefore PNGase free Endo F₁, F₂ and F₃ mixtures should be useful in the functional evaluation of the oligosaccharides in glycoproteins.