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Molecular dynamics simulations and the conformational mobility of blood group oligosaccharides.

Molecular dynamics simulations were carried out without explicit consideration of solvent to explore the conformational mobility of blood group A and H oligosaccharides. The potential energy force field of Rasmussen and co-workers was used with the CHARMM program on a number of disaccharide and trisaccharide models composed of fucose, galactose, glucose, N-acetyl glucosamine, and N-acetyl galactosamine chosen to represent various fragments of blood group oligosaccharides. In agreement with results of earlier studies, stable chair conformations were found for each pyranoside from which no transitions were detected in simulations as long as 800 ps. Exocyclic dihedral angles, including that of C5-C6, generally show numerous transitions on a time scale of approximately 5-30 ps. The dihedral angles of some but not all glycosidic linkages of blood group oligosaccharides show transitions on the time scale of 30-50 ps, implying that the extent of internal motion in blood group oligosaccharides depends strongly on linkage stereochemistry. For certain blood group A and H oligosaccharides that show limited internal motion in these simulations, we argue that the calculations are consistent with our previous analysis of 1H nuclear Overhauser enhancement (NOE) data that imply single conformations over a wide range of temperature and solvent conditions. While the trajectories are consistent with 13C T1 values that have been interpreted as indicating rigid conformations, measurements of 13C-NOE and T1 as a function of magnetic field strength are proposed as an improved method for experimental detection of the internal motion that is suggested for certain oligosaccharides in these simulations. The results of these simulations differ substantially from those of peptides of a similar molecular weight in that the oligosaccharides show much less internal motion.

Blood Group Antigens↗
PubMed1990

Determination of the conformation of Lewis blood group oligosaccharides by simulation of two-dimensional nuclear Overhauser data.

Through control of both the nmr probe temperature and of the solvent viscosity, phase-sensitive two-dimensional 1H nuclear Overhauser data (NOESY) at 300 and 500 MHz are obtained with excellent signal-to-noise ratios for Lewis blood group penta- and hexasaccharides isolated from human milk. Relatively long mixing times are required to produce measurable NOE intensities in these oligosaccharides, which makes a full relaxation matrix analysis necessary. By measurements of selective T1 for a few isolated 1H resonances, it was possible to generate a simulation of the complete NOESY spectrum at arbitrary mixing time for comparison with the experimental data. From an exhaustive search of the conformational space, it was found that only a small range of glycosidic dihedral angles of the nonreducing terminal Lewis blood group determinant fragments of the milk oligosaccharides LNF-2 and LND-1 produce simulated spectra agreeing within experimental error to the data. Conformational energy calculations reveal that each of these conformations is also one of minimum energy. It is concluded that the Lewis(a) and Lewis(b) oligosaccharides adopt relatively compact rigid structures in solution, as shown by the observation of cross peaks between protons in nonadjacent residues. Like the blood group A and H oligosaccharides, there exists only a small dependence of the conformation for Lewis(a) and Lewis(b) oligosaccharides on solvent. The apparent lack of dependence of conformation of these oligosaccharides on DMSO in D2O suggests that modification of solvent viscosity with mixtures of DMSO:D2O may provide a useful general strategy of NOESY studies of oligosaccharides.

Carbohydrate Conformation↗
PubMed2001

Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus.

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95 degrees C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.

Amino Acid Sequence↗
PubMed1995

Endoplasmic reticulum-to-cytosol transport of free polymannose oligosaccharides in permeabilized HepG2 cells.

Free polymannose oligosaccharides have recently been localized to both the vesicular and cytosolic compartments of HepG2 cells. Here we investigated the possibility that free oligosaccharides originating in the lumen of the endoplasmic reticulum (ER) are transported directly into the cystosol. Incubation of permeabilized cells in the absence of ATP at 37 degrees C led to the intravesicular accumulation of free Man9GlcNAc2 which was generated from dolichol-linked oligosaccharide in the ER. This oligosaccharide remained stable within the permeabilized cells unless ATP was added to the incubations at which time the Man9GlcNac2 was partially converted to Man8GlcNAc2, and both these components were released from an intravesicular compartment into the cytosolic compartment of permeabilized cells. In contrast, when permeabilized cells, primed with either free triglucosyl-oligosaccharide or a glycotripeptide, were incubated with ATP both these structures remained associated with the intravesicular compartment. As the conditions in which free oligosaccharides were transported out of the intravesicular compartment into the cytosolic compartment did not permit vesicular transport of glycoproteins from the ER to the Golgi apparatus our data demonstrate the presence of a transport process for the delivery of free polymannose oligosaccharides from the ER to the cytosol.

Adenosine Triphosphate↗
PubMed1988

Oligosaccharide heterogeneity of glycoproteins sulfated during the vegetative growth of Dictyostelium discoideum.

Macromolecules are sulfated during the vegetative growth of Dictyostelium discoideum. A characterisation of the structures of sulfated oligosaccharides associated with these macromolecules indicates that the oligosaccharides are heterogeneous. Endoglycosidase and pronase digestion were used with gel-filtration chromatography to obtain two different oligosaccharide fractions and a glycopeptide fraction; these were further characterised by ion-exchange and lectin-affinity chromatography and by acid hydrolysis. The data indicate that up to 43% of the sulfate is associated with typical N-linked oligosaccharides, that up to 5% is associated with N-linked oligosaccharides that are either very large or extremely highly charged, and that the remaining sulfate is associated with oligosaccharides non-N-linked to protein. Each fraction was also shown to be heterogeneous at most other structural levels. Electrophoretic analyses following the endoglycosidase and pronase treatments indicated that all of the macromolecules are glycoproteins and suggested further that at least two of the oligosaccharide fractions are located on different groups of glycoproteins.

Chromatography, Affinity↗
PubMed2004

Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis.

Negative ion nano-liquid chromatography/mass spectrometry (nano-LC/MS) and tandem mass spectrometry (nano-LC/MS(2)), using graphitised carbon as separating medium, were explored for analysing neutral and acidic O-linked and N-linked oligosaccharide alditols. Compared to the sensitivity of capillary LC/MS (flow rate of 6 microL/min) coupled with a conventional electrospray ionisation source, the nano-LC/MS (flow rate of 0.6 microL/min) with a nanoflow ion source was shown to increase the sensitivity ten-fold with a detection limit in the low-femtomole range. The absolute signals for the [M-nH](n-) ions of the oligosaccharides were increased 100-fold, enabling accumulation of high-quality fragmentation data in MS(2) mode, in which detection of low abundant sequence ions is necessary for characterisation of highly sialylated N-linked oligosaccharides. Oligosaccharides with high numbers of sialic acid residues gave dominant fragments arising from the loss of sialic acid, and less abundant fragments from cleavage of other glycosidic bonds. Enzymatic off-line desialylation of oligosaccharides in the low-femtomole range prior to MS(2) analysis was shown to increase the quality of the spectra. Automated glycofragment mass fingerprinting using the GlycosidIQ software confirmed the oligosaccharide sequence for both neutral desialylated as well as sialylated structures. Furthermore, the use of graphitised carbon nano-LC/MS enabled the detection of four sialylated O-linked oligosaccharides on membrane proteins from ovarian tissue (5 microg of total amount of protein).

Anions↗
PubMed2005

Structural characterization of 2-aminobenzamide-derivatized oligosaccharides using a matrix-assisted laser desorption/ionization two-stage time-of-flight tandem mass spectrometer.

Oligosaccharides were derivatized by reductive amination using 2-aminobenzamide (2-AB) and analyzed by matrix-assisted laser desorption/ionization two-stage time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS) in the positive ion mode. The major signals were obtained under these conditions from the [M+Na]+ ions for all 2-AB-derivatized oligosaccharides. A systematic study was conducted on a series of 2-AB-derivatized oligosaccharides to allow rationalization of the fragmentation processes. The MALDI-TOF/TOF-MS/MS spectra of the [M+Na]+ ions of 2-AB-derivatized oligosaccharides were dominated by glycosidic cleavages. These fragments originating both from the reducing and the non-reducing ends of the oligosaccharide yield information on sequence and branching. Moreover, the MALDI-TOF/TOF-MS/MS spectra were also characterized by abundant cross-ring fragments which are very informative on the linkages of the monosaccharide residues constituting these oligosaccharides. MALDI-TOF/TOF-MS/MS analysis of 2-AB-derivatized oligosaccharides, by providing structural information at the low-picomole level, appears to be a powerful tool for carbohydrate structural analysis.

Carbohydrate Sequence↗
PubMed1995

Recognition of type 1 chain oligosaccharides and lacto-series glycolipids by an antibody to human secretory component.

Binding of the mouse IgM antibody 6C4 is lost after treatment of human free secretory component with peptide N-glycosidase F (Bakos et al. (1991) J. Immunol. 146, 162-168) or periodate, suggesting that asparagine-linked oligosaccharides contain the epitope recognized by this antibody. Inhibition of antibody binding to free secretory component by milk oligosaccharides established that lacto-N-tetraose is the minimum structure recognized by the antibody, but larger oligosaccharides with terminal Gal beta 1-3GlcNAc sequences bind with much higher affinity. Antibody binding is enhanced by substitution with the Lewis Fuc alpha 1-4 and is inhibited by Fuc alpha 1-2Gal substitution. Free secretory component, however, does not bind other antibodies that recognize Le(a) or Leb oligosaccharides, and binding is lost after digestion with a beta-galactosidase that cleaves Gal beta 1-3 linkages but not after digestion with alpha-L-fucosidase. Therefore, the major epitope recognized by 6C4 on free secretory component is probably not an asparagine-linked Le(a) oligosaccharide. The antibody also binds to human milk lactoferrin, some human mucins, and lacto-series glycolipids including III4 alpha Fuc-lactotetraosyl ceramide and lactotetraosyl ceramide. Based on affinity chromatography of oligosaccharides released from free secretory component, the epitope recognized by antibody 6C4 is present on approximately 3.5% of the asparagine-linked oligosaccharides.

Animals↗
PubMed1994

Separation of pyridylamino oligosaccharides by high-performance liquid chromatography on an amine-bearing silica column.

Several neutral and sialylated pyridylamino (PA) oligosaccharides were separated on an amine-bearing silica column, PALPAK Type N. Neutral PA-oligosaccharides were fractionated according to the number of sugar residues by amine adsorption. Sialylated PA-oligosaccharides were separated by ion-exchange chromatography. An amine-bearing column was eluted with a mobile phase consisting of acetonitrile and water containing acetic acid titrated to pH 7.3 with triethylamine (TEAA buffer). A mixture of neutral and sialylated PA-oligosaccharides was separated by double-mode HPLC with a solvent program of decreasing acetonitrile concentration (70 to 51%) with a constant TEAA buffer concentration (0.03 M), and then an increasing TEAA buffer concentration (0.03 to 0.49 M) with a constant acetonitrile concentration (51%). This HPLC technique was applied to the comparative oligosaccharide pattern analysis of human Asn-linked oligosaccharides of normal and pathological IgG by hydrazinolysis. The result indicated clearly that oligosaccharides of IgG myeloma proteins have different core structures and ratios of sialic acid than those of IgG normal proteins.

Amines↗
PubMed1999

Core-branching pattern and sequence analysis of mannitol-terminating oligosaccharides by neoglycolipid technology.

The occurrence of mannitol-terminating oligosaccharides (2-substituted or 2,6-disubstituted) among the O-glycans released by alkaline borohydride treatment from glycoproteins of the nervous system has prompted the development of a microscale method to analyze the core-branching pattern and sequence by the neoglycolipid (NGL) technology, analogous to a method previously described for GalNAcol-terminating oligosaccharides (M. S. Stoll, E. F. Hounsell, A. M. Lawson, W. Chai, and T. Feizi, Eur. J. Biochem. 189, 499-507, 1990). The approach involves the selective cleavage at the core mannitol by mild periodate treatment and analysis of the reaction products as NGLs by in situ TLC/liquid secondary ion mass spectrometry. Oxidation conditions have been optimized using as reference compounds 2-, 3-, 4-, or 6-monosubstituted mannobi-itols, 3,6-disubstituted mannitol-terminating pentasaccharides, and 2-mono- and 2,6-disubstituted mannitol-terminating neutral and sialylated oligosaccharides isolated from brain glycopeptides. When a 2:1 molar ratio of periodate to alditol is used, the core mannitol is cleaved at the C3-C4 threo-diol bond and in the absence of a threo-diol cleavage occurs to a lesser extent at erythro-diols. Saccharide ring diols are not cleaved under these conditions, and it is also shown that the side chain of sialic acid on the oligosaccharide is largely unaffected. Substituents at 2- and 6-positions of the core mannitol can be identified, and the method is applicable to neutral and sialylated oligosaccharide alditols. Typically, the starting material is 5 nmol of oligosaccharide and 0.5-1 nmol of derivatives is applied for analysis. By this strategy, the core-branching pattern and position of sialic acid of two branched monosialylated mannitol-terminating oligosaccharide isomers have been determined.

Animals↗
PubMed1994

Structural analysis of novel rhamnose-branched oligosaccharides from the glycophosphosphingolipids of Leptomonas samueli.

Mild alkaline hydrolysis of the glycophosphosphingolipids of the protozoan Leptomonas samueli liberated several phosphoinositol-containing oligosaccharides (PI-oligosaccharides), which were purified by high performance anion exchange chromatography. The oligosaccharides in the resulting four fractions were characterized by methylation analysis, fast atom bombardment mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. The oligosaccharides contain the core structure Man alpha (1-4)GlcN alpha (1-6)-myo-inositol-1-OPO3, and are substituted with 2 mol of 2-aminoethylphosphonate per mol of oligosaccharide. The nonreducing ends of the oligosaccharides were terminated by rhamnose branched neutral and acidic xylose-containing penta-, hexa-, hepta- and octasaccharides, of which the three most abundant were shown to have the structures: [formula: see text] More tentative structures are also proposed for three minor oligosaccharides.

Animals↗
PubMed1996

Expression of L-PHA-binding proteins in breast cancer: reconstitution and molecular characterization of beta 1-6 branched oligosaccharides in three-dimensional cell culture.

Expression of beta 1-6 branched oligosaccharides in human breast cancer cells was investigated in vivo and in vitro. Lectin histochemical and lectin blotting analyses of surgically resected specimens were performed using L-PHA (phaseolus vulgaris leukoagglutinin) lectin, which binds to beta 1-6 oligosaccharides. The glycoproteins bearing beta 1-6 oligosaccharides of breast cancer tissues were found to be 170 kD and 120 kD in molecular weight, and the former appeared to be an epitope of carcinoembryonic antigen (CEA). The beta 1-6 oligosaccharides were expressed in both cancer cell lines at the outer layer of the colonies when cultured in type I collagen, but not in agarose gel. No correlation was observed between beta 1-6 expression and cell cycle. The beta 1-6 oligosaccharides did not coincide with breast cancer-associated antigens, such as CEA, MUC1, and cathepsin D. The beta 1-6 oligosaccharides of these cell lines were markedly inhibited when swainsonine, a mannosidase II inhibitor, was added to the culture medium. The 120 kD molecule, which was obtained from MCF-7 cells cultured in type I collagen gel, was consistent with that of breast cancer tissues and was similar to lysosome-associated membrane glycoproteins (LAMPs). The results suggest that the glycoproteins bearing beta 1-6 branched oligosaccharides in human breast cancer incorporate an epitope of CEA and human LAMPs and that the expression of LAMPs may depend on their surrounding matrices and may play an important role in cancer invasion or metastasis.

Breast Neoplasms↗
PubMed1993

Comparison of oligosaccharide processing among various insect cell lines expressing a secreted glycoprotein.

The processing of the N-linked oligosaccharide modifying a secreted alkaline phosphatase glycoprotein (SEAP) expressed with a recombinant Autographa californica nuclear polyhedrosis virus was evaluated in insect cell lines established from Spodoptera frugiperda, Trichoplusia ni, and Mamestra brassicae. Studies with Endoglycosidase H (Endo H), which removes high-mannose oligosaccharides, revealed that 79% of the intracellular SEAP produced in the M. brassicae-derived MB0503 cell line was Endo H resistant. The commonly used S. frugiperda Sf21 and Sf9 cell lines produced 44 and 21% Endo H-resistant intracellular SEAP, respectively. Detection of oligosaccharide moieties with lectins, which selectively recognize terminal sugars, identified only mannose residues on SEAP expressed in the six insect cell lines. However, the oligosaccharide moiety of SEAP expressed in a Chinese hamster ovary cell line contained sialic acid. Therefore, when expressed in mammalian cells, the oligosaccharide present on SEAP is processed into complex oligosaccharide, but in insect cells it is of the high-mannose type. Studies with inhibitors of the initial oligosaccharide processing steps demonstrated that all six cell lines possessed glycosidase I/II and mannosidase I activity and that glycosylation was required for secretion.

1-Deoxynojirimycin↗
PubMed2002

Detection of ligands for selectins in the oligosaccharide fraction of human milk.

BACKGROUND: Human milk contains a large variety of oligosaccharides which show struc tural similarities with ligands for selectins, a family of cell adhesion molecules which are involved in many cell-cell interactions. AIM OF THE STUDY: Due to their structural similarity with selectin ligands, human milk oligosaccharides were labelled with phosphatidyl ethanolamine to be able to investigate specific effects of antibodies against carbohydrate epitopes. METHODS: Various monoclonal antibodies against physiological selectin ligands were used to determine whether epitopes within human milk oligosaccharides are recognized. Oligosaccharides were isolated from human milk, transferred into neoglycolipids and separated using high performance thin layer chromatography prior to incubation with monoclonal antibodies for the selectin ligands sialyl-Lewis a (sLe a), sialyl-Lewis x (sLe x), Lewis x (Le x) and Lewis y (Le y) after Western blotting. Fast atom bombardment-mass spectrometry was then used to identify antibody-binding compounds. Results In the immunoassays all ligand epitopes except for Le y were detected in the oligosaccharide fraction of human milk. Anti-sLe a showed the most distinct reaction with N-acetylneuraminic acid containing neoglycolipids of which two were identified as neuraminylfucosyl-lacto-N-hexaose and neuraminyl-lacto-N-tetraose. Such oligosaccharides as well as similar structures are present in relatively high concentrations in human milk. CONCLUSIONS: The presence of sialyl-Lewis ligands on milk oligosaccharides together with their abundancy in human milk may suggest that they could be selectin ligands and they may be part of inflammatory processes.

Animals↗
PubMed1999

Alginate oligosaccharides stimulate VEGF-mediated growth and migration of human endothelial cells.

Alginate oligosaccharides cleaved from alginic acid polysaccharides of seaweed were tested to determine their ability to enhance proliferation and migration of human umbilical vein endothelial cells. A mixture of alginate oligosaccharides (5 microg/ml in culture broth) stimulated endothelial cell growth, [(3)H]thymidine uptake and migration in the presence of recombinant vascular endothelial growth factor 165 (VEGF(165)). In contrast, a high concentration mixture of the oligosaccharides ( approximately 100 microg/ml) suppressed cell growth. The stimulatory activity was comparable to that of heparin, with affinity to VEGF(165), and decreased on heparin-induced stimulation. Each effective oligosaccharide had guluronic acid at the reducing end. A mixture of alginate oligosaccharides (5 microg/ml) and the most paragraph signeffective fraction (1 microg/ml) stimulated endothelial cell migration. In the presence of VEGF and heparin, some alginate oligosaccharides with the peripheral guluronic acid demonstrated marked stimulatory effects, and one fraction also showed a migratory effect. These findings indicate novel activities of alginate oligosaccharide(s) in endothelial cell growth and migration and suggest synergistic and/or stabilizing effects on VEGF(165)-dependent stimulation of endothelial cells.

Alginates↗
PubMed1990

Lectin affinity chromatography of proteins bearing O-linked oligosaccharides: application of jacalin-agarose.

The lectin jacalin immobilized on agarose was found to bind a variety of glycoproteins known to contain typical O-linked oligosaccharides, including human IgA, C1 inhibitor, chorionic gonadotropin, plasminogen, bovine protein Z, bovine coagulation factor X, and fetuin. These proteins were eluted from columns of jacalin-agarose specifically by alpha-galactopyranosides such as melibiose and alpha-methylgalactopyranoside but not by lactose or other sugars. Treatment of asialofetuin with endo--alpha--N--acetylgalactosaminidase eliminated its affinity for the lectin column, and other proteins known to contain only N-linked oligosaccharides such as ovalbumin, transferrin, and alpha 1-acid glycoprotein were not retained by the lectin. Binding of proteins with O-linked oligosaccharides to the lectin column did not require divalent cations and was affected little by changes in pH and ionic strength over a wide range. Virtually all of the glycosidically linked oligosaccharides of fetuin, chorionic gonadotropin, and plasminogen are known to be sialated. Thus, binding of these glycoproteins to jacalin, which is known to have affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides of these proteins, was not prevented by the presence of sialic acids. Affinity of oligosaccharides for jacalin did appear to be reduced by occurrence of sialic acids as it was found that higher concentrations of melibiose were required to elute asialofetuin than fetuin from jacalin-agarose. Results of the present study indicate that affinity chromatography using this lectin is a widely applicable technique for identifying and purifying proteins bearing O-linked oligosaccharides.

Animals↗
PubMed1992

Analysis of oligosaccharides by on-line high-performance liquid chromatography and ion-spray mass spectrometry.

Oligosaccharides were analyzed by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). First, oligosaccharides labeled with 2-aminopyridine were studied to see if they could be analyzed by MS under the conditions used for separation by HPLC. Pyridylamino (PA)-oligosaccharides could be analyzed under these conditions, although the mass spectra were affected. Then, liquid chromatography-mass spectrometry was used to analyze a PA-oligosaccharide mixture derived from human immunoglobulin G. The PA-oligosaccharides were separated on a reversed-phase column and mass-analyzed directly. The observed molecular weights were close to or identical to those expected from the structures, which were estimated from the elution position on HPLC. This method is rapid and simple, as the mass spectrometer can give the accurate molecular weight of each PA-oligosaccharide in one chromatography run, even if the HPLC separation is incomplete. This method can be used to extend the so-called two-dimensional mapping of PA-oligosaccharides. The structure can be studied in greater detail by tandem MS.

Carbohydrate Conformation↗
PubMed1984

Oligosaccharides on cathepsin D from porcine spleen.

Cathepsin D from porcine spleen contained mannose (3.3%), glucosamine (1.4%), and mannose 6-phosphate (0.08%). Essentially all of the oligosaccharides of cathepsin D could be released by endo-beta-N-acetylglucosaminidase H, pointing to oligomannoside types of structures. Three neutral oligosaccharide fractions, containing 5, 6, and 7 mannose residues, respectively, were isolated by gel permeation chromatography on Bio-Gel P-2. Studies using exoglycosidase digestions and 500-MHz 1H NMR spectroscopy revealed that their structures are [Man alpha 1 leads to 2]0 or 1 Man alpha 1 leads to 6[Man alpha 1 leads to 3]Man alpha 1 leads to 6[Man alpha 1 leads to 2)0 or 1 Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4 GlcNAc. These structures are identical to what have recently been proposed by Takahashi et al. for the major oligosaccharide units of cathepsin D from the same source (T. Takahashi, P. G. Schimidt, and J. Tang (1983) J. Biol. Chem. 258, 2819-2930), except for the occurrence of two isomeric oligosaccharides containing six mannoses. Only a part (3.4%) of the oligosaccharides were acidic, containing phosphates in monoester linkage. The phosphorylated oligosaccharides also consisted of oligomannoside-type chains which were analogous to, but more heterogeneous in size than the neutral oligosaccharides. Cathepsin D was bound to a mannose- and N-acetylglucosamine-specific lectin (mannan-binding protein) isolated from rabbit liver with the Ki value of 5.4 X 10(-6) M.

Acetylglucosaminidase↗