Structures of the asparagine-linked sugar chains of laminin

This investigation describes the isolation and characterization of oligosaccharides of the basement membrane glycoprotein, laminin. Pronase-released glycopeptides of isolated laminin, from a mouse Engelbreth-Holm-Swarm tumor, were fractionated using a combination of gel permeation chromatography and Con A-Sepharose affinity chromatography. The glycopeptides were analyzed for sugar linkage patterns by methylation analysis. Glycopeptides and hydrazine-released oligosaccharides were further analyzed using endo-beta-galactosidase, endo-beta-N-acetylglucosaminidase H and specific exoglycosidases in conjunction with calibrated gel permeation chromatography. Based on these experiments, murine tumor laminin was shown to contain asparagine-linked oligosaccharides with the following structures: bi-, tri- and tetraantennary complex-type oligosaccharides; polylactosaminyl side chains containing Gal(beta 1----4)GlcNAc(beta 1----3) repeating units attached to the trimannose core portion of the bi-, tri- and tetraantennary complex-type oligosaccharides; unusual complex-type oligosaccharides terminated at the nonreducing end with sialic acid, alpha-galactose, beta-galactose and beta-N-acetylglucosamine; alpha-galactosyl residues linked to N-acetyllactosamine sequences; high-mannose-type oligosaccharides. These results, in conjunction with analytical data, indicate that most of the carbohydrate of this laminin is N-linked to asparagine and that there are about 43 such N-linked oligosaccharides per laminin molecule.     

Suppression of allogeneic reactivity in vitm by the syncytiotrophoblast membrane glycocalyx of the human term placenta is carbohydrate dependent

Immunosuppressive factors isolated from trophoblast are knownto block both innate and major histocompatability complex (MHC)-dependentcell-mediated immune responses in vitro and, in some cases,in vivo. We investigated the biochemical nature of these factors,which is presently unknown. Immunosuppressive activity, assessedby inhibition of two-way MLR, was extracted from term syncytiotrophoblastmicrovilli using 3 M KCl. The activity resisted both extensivepronase digestion and heating to 90°C for 1 h, demonstratingthat intact membrane proteins were not required. Although purifiedprotein-linked oligosaccharides released by hydrazinolysis fromthe syncytiotrophoblast membrane were themselves inactive, theyblocked the immunosuppressive activity of the KCl extract. Afterpronase digestion, the activity could be fractionated by TSK55S gel filtration, followed by C18 reverse-phase chromatography.Sequential exoglycosidase digestion of hydrazine-released sugarsof the active fraction demonstrated that it contained neutralN-linked oligomannose and hybrid oligosaccharides, which normallymake up <3% of the total syncytiotrophoblast-derived proteinglycan Library. These glycopeptides of the active fraction wereassociated with membrane phospholipid micelles. The possiblemechanism by which incompletely processed N-linked oligosaccharidesexpressed by a variety of syncytiotrophoblast membrane glycoproteinsmay block allogeneic reactivity when presented as polyvalentsugar groups is discussed. glycoprotein immunosuppression oligosaccharides pregnancy trophoblast     

Schistosoma mansoni synthesizes novel biantennary Asn-linked oligosaccharides containing terminal beta-linked N-acetylgalactosamine.

This report describes the structure of novel complex-type Asn-linked oligosaccharides in glycoproteins synthesized by the human blood fluke, Schistosoma mansoni. Adult schistosome worm pairs (male and female) isolated from infected hamsters were metabolically radiolabelled with either [3H]glucosamine, [3H]mannose or [3H]galactose. The glycopeptides prepared by pronase digestion of the total glycoprotein fraction were isolated by affinity chromatography on columns of immobilized Concanavalin A (Con A) and Wisteria floribunda agglutinin (WFA). A subset of glycopeptides, designated IIb, that bound to both Con A and WFA was isolated. WFA has been shown to have affinity for oligosaccharides containing beta 1,4-linked N-acetylgalactosamine (GalNAc) at their non-reducing termini. Compositional analysis of IIb glycopeptides demonstrated that they contained N-acetylglucosamine (GlcNAc), GalNAc, mannose (Man) and fucose (Fuc), but no galactose (Gal) or N-acetylneuraminic acid (NeuAc). Methylation analyses and exoglycosidase digestions indicated that IIb glycopeptides were complex-type biantennary structures with branches containing the sequence GalNAc beta 1-4-[+/- Fuc alpha 1-3]GlcNAc beta 1-2Man alpha 1-R. The discovery of these unusual oligosaccharides synthesized by a human parasite, which appear to be similar to some newly discovered mammalian cell-derived oligosaccharides, may shed light on future studies related to the role oligosaccharides may play in host-parasite interactions.     

Chemo-enzymatic synthesis of eel calcitonin glycosylated at two sites with the same and different carbohydrate structures

Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N-acetyl-D-glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo-beta-N-acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae. Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives.     

Carbohydrate-binding specificity of pokeweed mitogens

The carbohydrate-binding specificity of two pokeweed (Phytolacca americana) mitogens (Pa-1 and Pa-2) was investigated by means of hemagglutination inhibition assays and the quantitative inhibition of the binding of 125I-labeled lectins to human erythrocytes using various oligosaccharides, glycopeptides and glycoproteins as hapten inhibitors. Among the inhibitors employed in this study, chitin oligosaccharides and the glycopeptides and glycoproteins which bear sugar chains of the type found in serum glycoproteins, particularly PAS-1 glycoprotein and band-3 glycoprotein of human erythrocyte membranes, exerted strong inhibitory activity. The inhibitory constants of band-3 glycoprotein toward the binding of both mitogens to human erythrocytes were found to be very close to the association constants of the mitogens to the cells. Furthermore, the results of competitive binding studies between Pa-1 and Pa-2 indicated that these mitogens share a common oligosaccharide chains on the erythrocyte surface. To isolate the membrane receptors for these two mitogens, the solubilized membranes of human erythrocytes were subjected to affinity chromatographies using Pa-1-Sepharose 4B and Pa-2-Sepharose 4B as specific adsorbents. In both cases of these two specific adsorbents, band-3 glycoprotein was found to bind most strongly. These results suggest that two pokeweed mitogens have essentially the same carbohydrate-binding specificity and they bind primarily to the sugar chains of band-3 glycoprotein, possibly to the core structure of the sugar chains containing a di-N-acetylchitobiose moiety, on human erythrocytes.     

Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC.

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983).     

The oligosaccharide moieties of the epidermal growth factor receptor in A-431 cells. Presence of complex-type N-linked chains that contain terminal N-acetylgalactosamine residues

The receptor for epidermal growth factor (EGF) in the human epidermoid carcinoma cell line A-431 is a glycoprotein of apparent molecular weight = 170,000. During biosynthesis, the receptor is first detected as a precursor of apparent Mr = 160,000. In this report we describe our studies on the structures of the oligosaccharide moieties of the mature receptor and its precursor. A-431 cells were grown in medium containing radioactive sugars and the radiolabeled receptors were purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled glycopeptides were prepared from the purified receptor by proteolysis, and their structures were examined by a variety of techniques. The mature EGF receptor contains both complex-type and high mannose-type Asn-linked oligosaccharides in the approximate ratio of 2 to 1, while the precursor contains only high mannose-type chains. A number of experimental results demonstrate that the mature receptor does not contain oligosaccharides in O-linkage through N-acetylgalactosamine to either serine or threonine. The high mannose-type oligosaccharides in both precursor and mature receptor can be cleaved by endo-beta-N-acetylglucosaminidase H and occur in the mature receptor as Man9GlcNAc2 (6%), Man8GlcNAc2 (49%), Man7GlcNAc2 (25%), and Man6GlcNAc2 (20%), whereas, in the receptor precursor the high mannose chains occur primarily as Man8GlcNAc2 (70%). The complex-type oligosaccharides in the mature receptor are predominantly tri- or tetraantennary species and are unusual in several respects. (i) Many of the chains do not contain sialic acid, while the remaining chains contain 1-2 sialic acid residues. (ii) Half of the [3H] mannose-derived radioactivity was recovered as [3H] fucose and the remaining half as [3H] mannose, indicating that there may be an average of 3 fucose residues/chain. (iii) About one-third of the [3H] glucosamine-derived radioactivity in these glycopeptides was recovered as N-acetylgalactosamine and these residues are all alpha-linked and occur at the nonreducing termini. These data demonstrate that the complex-type Asn-linked oligosaccharides in the EGF receptor from A-431 cells contain sugar residues related to human blood type A. In light of other recent studies, these results suggest that in A-431 cells blood group determinants in surface glycoproteins are contained in Asn-linked but not O-linked oligosaccharides.     

Carbohydrates of human immunodeficiency virus. Structures of oligosaccharides linked to the envelope glycoprotein 120

Human T-cells (H9), persistently infected with the HTLV-III strain of human immunodeficiency virus, were metabolically labeled with D-[2-3H]mannose or D-[6-3H]glucosamine. The viral envelope glycoprotein, gp120, was isolated either from cell lysates or from cell-free culture supernatant. After proteolytic digestion, the radiolabeled oligosaccharides were sequentially liberated from glycopeptides by treatment with endo-beta-N-acetylhexosaminidase H and peptide:N-glycosidase F. Oligosaccharides released were separated from residual (glyco)peptides and fractionated according to size, charge, and fucose content. The individual oligosaccharide species obtained were characterized by digestion with exoglycosidases and by chromatographic comparison with standard oligosaccharides. Our results demonstrate that the intracellular gp120 carries predominantly oligomannosidic glycans comprising nine or eight mannose residues. The secreted glycoprotein is equally substituted by oligomannosidic species, containing seven to nine mannose residues, and by fucosylated, partially sialylated bi- and triantennary complex-type oligosaccharides.     

Synthesis of 1-N-glycyl beta-oligosaccharide derivatives. Reactivity of Lens culinaris lectin with a fluorescent labeled streptavidin pseudoglycoprotein and immobilized neoglycolipid.

The lectin from Lens culinaris (lentil) has a binding specificity for glycopeptides bearing 6-O-linked fucose on the reducing terminus on complex-type N-linked oligosaccharides. Lentil lectin therefore provides an excellent example of a carbohydrate binding protein in which high-affinity interactions are dependent on the integrity of the oligosaccharide core structure. We report here the synthesis of the 1-N-glycyl beta-derivative of Gal beta 4GlcNAc beta 2Man alpha 6(Gal beta 4GlcNAc beta 2Man alpha 3)Man beta 4GlcNAc beta 4(Fuc alpha 6)-GlcNAc (Gal-2F) and its subsequent biotinylation and palmitoylation. The biotin derivative when bound to a streptavidin-fluorescein isothiocyanate (FITC) conjugate was able to bind to both concanavalin A (ConA) and lentil lectin affinity columns. In contrast, synthesis of the biotin derivative of the glycamine derivative of Gal-2F and subsequent binding to streptavidin-FITC afforded reactivity to a ConA affinity column but not to a lentil lectin affinity column. Lentil lectin also bound to plastic microtiter plates containing the adsorbed palmitoyl-1-N-glycyl beta-derivative. No binding occurred when the homologous glycamine neoglycolipid was used. These results suggest the 1-N-glycyl beta-derivative of oligosaccharides may have general utility as an intermediate in the synthesis of novel glycoconjugate probes.     

Natural killer cells discriminate between high mannose- and complex-type asparagine-linked oligosaccharides

Chinese hamster ovary cell lines with different types of N-linked oligosaccharides were tested as targets for control and lymphokine treated natural killer (NK) cells. The targets tested were parent cells, Lec1 mutants and Lec4 mutants. Due to an apparent defect in GlcNAc transferase V, Lec4 cells produce complex-type N-linked oligosaccharides devoid of GlcNAc beta(1-6) linked branches. Lec1 cells form only high mannose-type N-linked oligosaccharides because they lack GlcNAc transferase I activity. Lec1 cells are very sensitive to lysis by beta-interferon treated human NK cells, but both parent and Lec4 cells are resistant to NK lysis. The ability to discriminate between parent and Lec1 targets was demonstrated with untreated control effectors as well as those which were pretreated with either beta-interferon, gamma-interferon or interleukin-2. Both control and lymphokine-boosted NK cells exhibit much greater lytic activity against targets having only high mannose-type N-linked oligosaccharides. Five oligosaccharide structures resembling those found on N-linked glycoproteins were tested for their ability to block NK lysis of Lec1 targets. Only the high mannose-type glycopeptide from 7S soybean glycoprotein was inhibitory in the mu molar range. At the same concentration, none of the complex-type oligosaccharides had any effect on lytic activity. The results suggest that a high mannose-type N-linked oligosaccharides is recognized at some step in NK cell-mediated lysis.     

Specificity of concanavalin A binding to asparagine-linked glycopeptides. A nuclear magnetic relaxation dispersion study

We have investigated the binding of a series of high affinity asparagine-linked glycopeptides, including high mannose type and a bisected hybrid type, and several related synthetic oligosaccharides, to Ca2+- Mn2+-concanavalin A (ConA), using solvent proton nuclear relaxation dispersion (NMRD) measurements. We find that binding of the glycopeptides induces a common smaller decrease in the NMRD profile of ConA compared to that induced by monosaccharide binding. This effect is also observed with a synthetic analog of complex-type carbohydrates, hepta, which also shows enhanced affinity for the protein relative to monosaccharide binding. The high affinity of the glycopeptides and hepta, and their unique effects on the NMRD profile, are mimicked by binding of the trimannosyl oligosaccharide, 3,6-di-O-(alpha-D-mannopyranosyl)-D-mannose, which is present as a structural element in all of the glycopeptides and synthetic oligosaccharides. However, adding a so-called bisecting N-acetyl-D-glucosamine residue to the trimannosyl oligosaccharide greatly reduces its binding affinity and produces a decrease in the NMRD profile of the protein similar to that observed for monosaccharide binding. These results indicate that the trimannosyl oligosaccharide is a unique moiety recognized by the lectin for high affinity and extended site binding, and the presence of a bisecting N-acetyl-D-glucosamine residue in the trimannosyl oligosaccharide eliminates this type of interaction. The results also demonstrate that ConA primarily binds to the outer trimannosyl regions of high mannose and bisected hybrid-type glycopeptides compared to the central trimannosyl region of complex glycopeptides. Two mechanisms of enhanced affinity binding of saccharides and glycopeptides to ConA are discussed.     

Biosynthesis of glycoconjugates in mitochondrial outer membranes. Preliminary characterization of the oligosaccharide moiety of a N-glycoprotein

Outer mitochondrial membranes synthesize a N-glycoprotein by a direct incorporation of sugars from their nucleotide-donors into an endogenous protein acceptor. To characterize the oligosaccharide moiety of this N-glycoprotein, we sequentially incorporated [14C]-sugars into the protein acceptor. After pronase digest, the released [14C]-glycopeptides were fractionated on a QAE-Sephadex column which gave rise to 9% of neutral glycopeptides and 91% of charged glycopeptides. These latter were identified as bearing phosphate residues in the form of monoester (28%) and acid-stable diester (63%). The oligosaccharide moiety of this mitochondrial glycoprotein has been characterized as incomplete biantennary complex-type chains.     

Screening for complex carbohydrates inhibiting hemagglutinations by CFA/I- and CFA/II-expressing enterotoxigenic Escherichia coli strains

Abstract Numerous structural families of naturally occurring glycopeptides and oligosaccharides have been evaluated as potential inhibitors of hemagglutinations mediated by CFA/I- and CFA/II-positive enterotoxigenic Escherichia coli strains. Among the preparations tested were glycopeptides with short O-linked (mucin-type) chains, various mixtures containing N-linked glycans (either oligomannoside-, hybrid- or complex-type), three fractions of human milk oligosaccharides, and glycopeptides derived from either pooled new-born meconiums or pooled human red blood cell membranes. In almost all cases, the same inhibitory preparations were active toward all E. coli strains. This emphasizes the close analogy between the carbohydrate specificities of the colonization factors concerned. Such inhibitors always contained lactosamine units in their oligosaccharide backbones, but this structural requirement alone was not sufficient for activity. The glycopeptide mixture derived from human erythrocyte membranes (known to contain blood group-related carbohydrate antigens carried by a lactosaminoglycan backbone) behaved as a potent hemagglutination inhibitor, especially towards CFA/II-expressing strains. This last result clearly indicates the structural family in which complex carbohydrates should be selected to establish precisely the specificity of these CFA/II adhesins.     

Sertoli cell glycosylation patterns as affected by culture age and extracellular matrix

This study evaluated the responsiveness of Sertoli cell glycosylation in vitro to changes in culture age and to the presence of a reconstituted basement membrane (Matrigel) or collagen IV/laminin substrata. Primary Sertoli cell cultures were prepared from 20-day-old rats and incubated with [3H]mannose, a monosaccharide specific for asparagine-linked oligosaccharides. The cells were harvested on Days 4, 6, or 10 of culture life. A supernatant enriched in cell-surface glycopeptides (the trypsinate) and a cell pellet stripped of surface glycoconjugates were evaluated separately. Glycopeptides derived from a Pronase digest of the two samples were fractionated using concanavalin-A lectin affinity chromatography into three major classes: multiantennary complex-type, biantennary complex-type, and high-mannose-type oligosaccharide structures. The proportion of radiolabeled glycopeptides appearing in each of the three classes did not differ between Days 4 and 6 of culture. In contrast, a significant increase in the percentage of radiolabeled glycopeptides containing multiantennary complex-type oligosaccharides was observed in cells harvested from the 10-day-old cultures. In other experiments, Sertoli cells were grown on various substrata: plastic; collagen IV/laminin; or Matrigel, a reconstituted basement membrane (RBM) composed of laminin, collagen IV, proteoglycan sulfate, entactin, and nidogen. Growth on RBM significantly increased multiantennary complex-type oligosaccharide formation compared to plastic, whereas the high-mannose-type glycopeptides increased in cells grown on collagen IV/laminin. These studies suggest that environmental and physiological conditions such as culture age and the presence of extracellular matrix significantly affect glycosylation patterns in Sertoli cell cultures.     

Requirement of the core structure of a complex-type glycopeptide for the binding to immobilized lentil- and pea-lectins

Structural requirements for the binding of oligosaccharides and glycopeptides to immobilized lentil- and pea-lectins were investigated by use of radioactively-labeled glycopeptides and oligosaccharides. The results indicate that an intact 2- acetamido-2-deoxy-β-d-glucopyranosyl residue at the reducing end of a complex-type oligosaccharide is essential for high-affinity binding to lentil lectin-Sepharose but not to concanavalin A-Sepharose and that an asparagine residue is required for the binding of a complex-type glycopeptide to pea lectin-Sepharose. In addition, interaction of a complex-type oligosaccharide with lentil lectin-Sepharose was enhanced by exposure of nonreducing, terminal 2-acetamido-2-deoxy-β-d-glucopyranosyl groups, whereas interaction with pea lectin-Sepharose was enhanced only after exposure of nonreducing, terminal α-d-mannopyranosyl groups.     

Age-Dependent Changes in the Oligosaccharide Structure of the Major Myelin Glycoprotein, P0

The single oligosaccharide moiety of the major myelin glycoprotein, P0, resides in an immunoglobulin-like domain that appears to participate in homophilic binding. The studies presented here indicate that the structure of the P0 oligosaccharide from rat nerve changes as a function of Schwann cell age. Examination of 5-day-old nerve revealed that P0 contained predominantly endo-beta-N-acetylglucosaminidase H (endo H)-resistant, complex-type oligosaccharide. In contrast, P0 from adult rats had mostly endo H-sensitive carbohydrate, indicating the presence of appreciable high-mannose and/or hybrid-type oligosaccharide on the glycoprotein. The endo H-sensitive and -resistant P0 of adult nerve could be readily phosphorylated by protein kinase C, as could the complex-type P0 from 5-day-old nerve. This suggests that the glycoprotein progresses to the plasma membrane and myelin regardless of the type of oligosaccharide chain. Analysis of 35SO4(2-)-labeled P0 showed that the sulfate group was found on both endo H-sensitive and -resistant oligosaccharide. The endo H-sensitive P0 carbohydrate from adult nerve appears to be primarily of the hybrid type, as evidenced by (a) the elution profile of [3H]mannose-labeled P0 glycopeptides from adult nerve during concanavalin A chromatography and (b) the inability of P0 from adult nerve to interact with Galanthus nivalis agglutinin. The observed age-dependent changes of P0 oligosaccharide may modify the binding properties of this myelin glycoprotein.     

High-mannose oligosaccharides from human Tamm-Horsfall glycoprotein

The present communication reports the occurrence of high-mannose oligosaccharides on Tamm-Horsfall glycoprotein prepared from human pooled urine. The Pronase digest of the glycoprotein was fractionated by gel filtration and a high-mannose glycopeptide species was separated from complex-type glycopeptides. When high-mannose glycopeptides were digested with endo--N-acetylglucosaminidase H, followed by reduction with [³H]KBH₄, three oligosaccharides were resolved by thin-layer chromatography. On the basis of chromatographic mobility and exoglycosidase digestions the composition Man₇-, Man₆-, and Man₅-GlcNAc was assigned to the three oligosaccharides. Man₆GlcNAc is by far the major component.     

Synthesis of the major oligosaccharide components of murine haemangioendothelioma cells

Murine haemangioendothelioma cells in culture synthesize lactosaminoglycan-type glycoproteins which are found both associated with cells and secreted into the culture medium. Pronase-derived glycopeptides, prepared from the [3H]glucosamine-labeled glycoproteins that were secreted into the culture medium were found to contain about 10% of the labeled products as large size (Mr greater than 5000) 3H-labeled glycopeptides. In contrast, 40% of the cellular 3H-glycopeptides were found to be of this large size class of glycopeptides. These large size glycopeptides did not bind to Con A-Sepharose but did bind to Datura stramonium-agarose, from which they were eluted with chitobiose. The glycopeptides which did not bind to Datura-lectin were sulfated complex-type oligosaccharides which were not degraded by endo-beta-galactosidase. The glycopeptides which bound to Datura-lectin were degraded by endo-beta-galactosidase (or keratanase) to yield Gal----GlcNAc----Gal and glycopeptides, which were resistant to further endo-beta-galactosidase digestion and which no longer bound to Datura lectin-agarose. A major [3H]glucosamine-labelled glycoprotein (Mr approx. 75000) was found to be susceptible to endo-beta-galactosidase degradation and is probably the major cellular constituent having lactosaminoglycan-type side chains in these cells. An in vitro assay to measure leucocyte-haemangioendothelioma interactions indicated that treatment of haemangioendothelioma cells with endo-beta-galactosidase reduced leucocyte binding to these cells by 80%.     

Analysis of site-specific glycosylation in recombinant human follistatin expressed in Chinese hamster ovary cells.

Follistatin (FS), a glycoprotein, plays an important role in cell growth and differentiation through the neutralization of the biological activities of activins. In this study, we analyzed the glycosylation of recombinant human FS (rhFS) produced in Chinese hamster ovary cells. The results of SDS-PAGE and MALDI-TOF MS revealed the presence of both non-glycosylated and glycosylated forms. FS contains two potential N-glycosylation sites, Asn95 and Asn259. Using mass spectrometric peptide/glycopeptide mapping and precursor-ion scanning, we found that both N-glycosylation sites were partially glycosylated. Monosaccharide composition analyses suggested the linkages of fucosylated bi- and triantennary complex-type oligosaccharides on rhFS. This finding was supported by mass spectrometric oligosaccharide profiling, in which the m/z values and elution times of some of the oligosaccharides from rhFS were in good agreement with those of standard oligosaccharides. Site-specific glycosylation was deduced on the basis of the mass spectra of the glycopeptides. It was suggested that biantennary oligosaccharides are major oligosaccharides located at both Asn95 and Asn259, whereas the triantennary structures are present mainly at Asn95.     

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.