Characterization of a glycopeptide from pathological human IgM with an unusual oligosaccharide core.

A glycopeptide from human pathological IgM has been prepared and characterized. It contains an unusual oligosaccharide core with only one N-acetylglucosamine carrying six mannosyl residues, and linked to the asparagine. The involvement of sugar has been determined by use of α and β mannosidases and endoglycosidase digestions. The amino acid sequence has been elucidated and from the obtained data it could be located on the C terminal portion of the IgM Du heavy chain at the Asn 563.This glycopeptide is characterized by the absence of a N-N-diacetylchitobiose unit in the oligosaccharide moiety and its condensed structure is proposed:

     

Some peculiarities of the dynamics of the immunoglobulin M structure

The isotropic mobility of separate regions of the intact molecule of immunoglobulin M (IgM) and its Fab and (Fc)5 fragments was studied using spin-labeling of carbohydrate (2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl) and peptide (2,2,5,5-tetramethyl-3-dichlorotriazinylaminopyrrolidine-1-oxyl) moieties. The spin-labeled oligosaccharide groups (OGs) in the Fab region are shown to have much more amplitude of anisotropic motion than those in the (Fc)5 region. The spin label in the latter is evidently attached in the C mu 3 domain to one of its OGs which is probably stabilized by ionic contacts between terminal N-acetylneuraminic acid residue and the peptide moiety of the IgM molecule. When the amount of the glycosidase-cleaved carbohydrate does not exceed 10-15%, most OGs affected are of the Fab region. Upon profound splitting (greater than or equal to 50%) the OGs of the (Fc)5 region are also affected; that results evidently in loosening the ionic contacts between the shortened OGs and the peptide moiety of IgM, and consequently in increasing mobility of the former. The structure of the (Fc)5 region of IgM is labile; after detaching this moiety from the intact IgM molecule, its structure is stabilized, but one of its domains (C mu 3) becomes more mobile than it is in the intact IgM molecule; at the same time the amplitude of anisotropic motion of OG bound here is decreased. In the latter case, this decrease depends on the sequence of spin-labeling and fragmentation. The most probable cause of stabilization of the (Fc)5 fragment is the heating of IgM solution to 56 degree C during fragmentation with trypsin. At this temperature the tau value for the (Fc)5 fragment is unusually low, equaling 23 ns. The spin-labeling in the peptide part of IgM occurs mostly in the Fab region which is a rather rigid moiety as expected.     

Some Peculiarities of the Dynamics of the Immunoglobulin M Structure

Abstract

The isotropic mobility of separate regions of the intact molecule of immunoglobulin M (IgM) and its Fab and (Fc)₅ fragments was studied using spin-labeling of carbohydrate (2,2,6,6-tetramethyl-4-aminopiperidine-l-oxyl) and peptide (2,2,5,5-tetramethyl-3-dichloro-triazinylaminopyrrolidine-l-oxyl) moieties.

The spin-labeled oligosaccharide groups (OGs) in the Fab region are shown to have much more amplitude of anisotropic motion than those in the (Fc)₅ region. The spin label in the latter is evidently attached in the Cμ3 domain to one of its OGs which is probably stabilized by ionic contacts between terminal N-acetylneuraminic acid residue and the peptide moiety of the IgM molecule.

When the amount of the glycosidase-cleaved carbohydrate does not exceed 10–15%, most OGs affected are of the Fab region. Upon profound splitting (≥50%) the OGs of the (Fc)₅region are also affected; that results evidently in loosening the ionic contacts between the shortened OGs and the peptide moiety of IgM, and consequently in increasing mobility of the former.

The structure of the (Fc)₅ region of IgM is labile; after detaching this moiety from the intact IgM molecule, its structure is stabilized, but one of its domains (Cμ3) becomes more mobile than it is in the intact IgM molecule; at the same time the amplitude of anisotropic motion of OG bound here is decreased. In the latter case, this decrease depends on the sequence of spin-labeling and fragmentation.

The most probable cause of stabilization of the (Fc)₅ fragment is the heating of IgM solution to 56°C during fragmentation with trypsin. At this temperature the τ value for the (Fc)₅ fragment is unusually low, equaling 23 ns.

The spin-labeling in the peptide part of IgM occurs mostly in the Fab region which is a rather rigid moiety as expected.     

The effect of peptide deletions on the glycosylation of murine immunoglobulin M heavy chains

The glycosylation and processing of the asparagine-linked oligosaccharides at individual glycosylation sites on the mu-chain of murine immunoglobulin M were investigated using variant cell lines that synthesize and secrete IgM heavy chains with known peptide deletions. Normal murine IgM has five N-linked oligosaccharides in the constant region of each heavy or mu-chain. Each mu-chain has four complex-type oligosaccharides as well as a single high mannose-type oligosaccharide near the carboxyl terminus of the molecule. The peptide deletion of the C mu 1 constant region domain in the heavy chains synthesized by one variant cell line did not prevent subsequent glycosylation at more distal glycosylation sites. In fact, the presence of this deletion resulted in more complete glycosylation at the C-terminal glycosylation site. Evaluation of glycopeptides containing individual glycosylation sites by Concanavalin A-Sepharose indicated that this deletion had no significant effect on the processing of structures from high mannose-type to complex-type oligosaccharide chains. In contrast, a deletion of the C-terminal peptide region of the heavy chain of IgM synthesized by a second variant cell line resulted in intracellular processing to more highly branched oligosaccharide structures at several of the glycosylation sites not involved in the deletion.     

D-galactofuranose in the N-linked sugar chain of a glycopeptide from Ascobolus furfuraceus

Two types of linkages between the carbohydrate and the peptide moiety in the glycopeptide from Ascobolus furfuraceus are described. Treatment with mild alkali produced beta-elimination of a small oligosaccharide. Evidence for the O-glycosidic linkage was provided by increase in absorbance at 240 nm, decrease in threonine and serine content after the alkaline treatment and detection of tritiated oligosaccharide following alkaline NaB3H4 reduction. Mannose is the sugar involved in the O-glycosidic linkage. The remaining glycopeptide was branched by galactofuranose units, which were selectivity released by mild acid hydrolysis. The N-glycosidic linkage of the sugar chain was conclusively proved by cleavage with endo-beta-N-acetyl-glucosaminidase. Sequential NaB3H4 reduction and acid hydrolysis gave [3H]glucosaminitol. The structure of the sugar chain was studied by 13C NMR spectroscopy and by methylation analysis.     

Structural characterization of the oligosaccharides of a human monoclonal anti-lipopolysaccharide immunoglobulin M

The oligosaccharide side chains of a human anti-lipopolysaccharide IgM produced by a human-human-mouse heterohybridoma were analyzed at each of its five conserved N-glycosylation sites. This antibody also has a potential sixth N-glycosylation site in the variable region of its heavy chain which is not glycosylated. The oligosaccharides were released by digestion with various endo- and exoglycosidases and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and fluorophore-assisted carbohydrate electrophoresis. The antibody has various complex- and hybrid-type oligosaccharide structures at Asn 171, various sialylated complex-type oligosaccharides at Asn 332 and 395, and high-mannose-type oligosaccharides at Asn 402 and 563. Of note is the presence in this human IgM of oligosaccharides containing N-glycolylneuraminic acid and N-acetylneuraminic acid in the ratio of 98:2 as determined using anion- exchange chromatography. Furthermore, we observed oligosaccharide structures containing Gal alpha (1,3)Gal that have not been reported as components of human glycoproteins.      

A lipid-linked oligosaccharide intermediate in glycoprotein synthesis in oviduct. Structural studies on the oligosaccharide chain.

The structure of the oligosaccharide chain of the lipid-linked oligosaccharide that serves as a donor of oligosaccharide chain to proteins of hen oviduct membranes has been investigated. A [Man-14C]glycopeptide fraction was prepared from membrane glycoproteins labeled with GDP-[14C]mannose. Reductive alkaline cleavage of this glycopeptide yielded a reduced oligosaccharide that, by four criteria, was identical with reduced [Man-14C]oligosaccharide prepared from [Man-14C]oligosaccharide-lipid. The structure of the oligosaccharide chain of the [Man-14C]glycopeptide was investigated by cleavage with a specific endo-beta-N-acetylglucosaminidase, followed by treatment of the released oligosaccharide with purified al alpha-and beta-mannosidases. By this procedure it was possible to establish the structure of the cleavage product as (alpha-Man)n-beta-Man-(1 leads to 4)-GlcNAc. Similar studies were performed on the [GlcNAc-14C]oligosaccharide prepared by hydrolysis of [GlcNAc-14C]oligosaccharide-lipid. The results indicate that the structure of the intact oligosaccharide is (alpha-Man)n-beta-Man-(1 leads 4)-beta-GlcNAc-(1 leads to 4)-GlcNAc. These experiments, coupled with earlier enzymatic studies on synthesis of the glycoproteins from the lipid-linked oligosaccharide, provide strong evidence that the structure of the oligosaccharide intermediate and the oligosaccharide chain of the glycoprotein product contain the same core structure found in many secretory glycoproteins.     

Structure of the high mannose oligosaccharides of a human IgM myeloma protein. I. The major oligosaccharides of the two high mannose glycopeptides.

The structures of the predominant high mannose oligosaccharides present in a human IgM myeloma protein (Patient Wa) have been determined. The IgM glycopeptides, produced by pronase digestion, were fractionated on DEAE-cellulonalysis shows that glycopeptide I contains Asn, Pro, Ala, Thr, and His and glycopeptide II contains Asn, Val, and Ser, which are the same amino acids found in the sequences around Asn 402 and Asn 563 respectively, to which high mannose oligosaccharides are attached in IgM (Patient Ou) (Putnman, F.W., Florent, G., Paul, C., Shinoda, T., and Shimizu, A. (1973) Science 182, 287-290). The high mannose glycopeptides in IgM (Wa) exhibit heterogeneity in the oligosaccharide portion. Structural analysis of the major oligosaccharides indicates that the simplest structure is: (see article of journal). The larger oligosaccharides present have additional mannose residues linked alpha 1 yields 2 to terminal mannose residues in the above structure. Glycopeptide I contains primarily Man5 and Man6 species, while glycopeptide II contains Man6 and Man8 species. The two Man6 oligosaccharides have different branching patterns.     

Structure of glycoproteins of human erythrocytes. Alkali-stable oligosaccharides

Studies have been made on the oligosaccharide residues of the alkali-stable carbohydrate-protein linkage of sialoglycopeptides derived from human erythrocytes. Four glycopeptides were isolated after alkaline borohydride treatment and Pronase digestion of MN-active sialoglycopeptides. The structure of one of these glycopeptides (GPIV) has been studied by sequential hydrolysis with specific glycosidases. Glycopeptide GPIV contained (per mol): 1mol of fucose, 1mol of sialic acid, 3mol of galactose, 3mol of mannose, 4mol of acetylglucosamine, 1mol of aspartic acid and fractional amounts of threonine, serine and glycine. The molecular weight of the glycopeptide was estimated to be 2330 by gel filtration. On the basis of glycosidase-digestion results, a tentative structure is proposed for the oligosaccharide moiety of glycopeptide GPIV.     

Demonstration of peptide:N-glycosidase F activity in endo-beta-N-acetylglucosaminidase F preparations

Endo-beta-N-acetylglucosaminidase F preparations from Flavobacterium meningosepticum have been found to contain peptide:N-glycosidase activity. Only the second activity, designated as peptide:N-glycosidase F, readily cleaves the beta-aspartylglycosylamine linkage of a fetuin triantennary complex glycopeptide, as shown by the isolation of the corresponding carbohydrate-free peptide containing aspartic acid and of an intact oligosaccharide with a di-N-acetylchitobiosyl moiety at the reducing end. Both activities in the mixture will hydrolyze a high mannose octaglycopeptide from ovalbumin, with the type of product formed being influenced by pH. At pH 4.0, only the endo-beta-N-acetylglucosaminidase F activity is functional, releasing octapeptide-GlcNAc and oligosaccharide-GlcNAc. At pH 9.3, the predominant cleavage is by peptide:N-glycosidase F at the glycosylamine bond, releasing octapeptide and oligosaccharide-GlcNAc-GlcNAc. This latter oligosaccharide is then hydrolyzed by endo-beta-N-acetylglucosaminidase F to oligosaccharide-GlcNAc plus GlcNAc.     

Major carbohydrate structures at five glycosylation sites on murine IgM determined by high resolution 1H-NMR spectroscopy

Mouse myeloma immunoglobulin IgM heavy chains were cleaved with cyanogen bromide into nine peptide fragments, four of which contain asparagine-linked glycosylation. Three glycopeptides contain a single site, including Asn 171, 402, and 563 in the intact heavy chain. Another glycopeptide contains two sites at Asn 332 and 364. The carbohydrate containing fragments were treated with Pronase and fractionated by elution through Bio-Gel P-6. The major glycopeptides from each site were analyzed by 500 MHz 1H-NMR and the carbohydrate compositions determined by gas-liquid chromatography. The oligosaccharide located at Asn 171 is a biantennary complex and is highly sialylated. The amount of sialic acid varies, and some oligosaccharides contain alpha 1,3-galactose linked to the terminal beta 1,4-galactose. The oligosaccharides at Asn 332, Asn 364, an Asn 402 are all triantennary and are nearly completely sialylated on two branches and partially sialylated on the triantennary branch linked beta 1,4 to the core mannose. The latter is sialylated about 40% of the time for all three glycosylation sites. The major oligosaccharide located at Asn 563 is of the high mannose type. The 1H-NMR determination of structures at Asn 563 suggests that the high mannose oligosaccharide contains only three mannose residues.     

Oligosaccharide units of lysosomal cathepsin D from porcine spleen. Amino acid sequence and carbohydrate structure of the glycopeptides

The amino acid sequences near the glycosylation sites and the oligosaccharide structures have been determined for the lysosomal protease cathepsin D from porcine spleen. Cathepsin D light and heavy chains were separately digested with proteases and the glycopeptides were purified. A single sequence was constructed from the amino acid sequence of the light chain glycopeptides which is: Tyr-Asn-Ser-Gly-Lys-Ser-Ser-Thr-Tyr-Val-Lys-Asn(CH2O)-Gly-Thr-Thr-Phe. A single glycopeptide sequence was also obtained for the heavy chain: Lys-Gly-Ser-Leu-Asp-Tyr-His-Asn(CH2O)-Val-Thr-Arg-Lys-Ala-Tyr. The light chain sequence is homologous with the sequence of porcine pepsin from residues 56 to 71. The heavy chain sequence is homologous with the pepsin sequence from residues 176 to 189. Thus, the 2 oligosaccharide-linked asparagines in cathepsin D correspond to residues 67 and 183 in pepsin and other homologous aspartyl proteases. These positions are located on the surface of the crystal structures of aspartyl proteases. Five oligosaccharides linked to Asn-67 were separated and their structures determined with proton NMR. Four major oligosaccharides are structural variants from the high mannose-type having 3, 5, 6, and 7 mannoses, respectively. A minor structure contained a third GlcNAc. Three oligosaccharide structures were found linked to Asn-183. Two major oligosaccharides are of the high mannose-type each with 5 mannose residues. One of the two contains a fucose linked to a GlcNAc. A third, very minor oligosaccharide contains galactose.     

Synthesis of the glycosylated polypeptide chain of an inducible costimulator on T-cells

The glycoprotein AILIM/ICOS (Activation inducible lymphocyte immunomediately molecule/Inducible co-stimulator) on T-cells was identified in 1998 as a member of the CD28/CTLA4 family. The three-dimensional structure of the AILIM/ICOS extracellular domain has not been solved, and therefore we have examined the preparation of homogeneous glycosylated polypeptide chains of this domain having two homogeneous N-linked complex type oligosaccharides for use in folding experiments. To synthesize the glycosylated whole polypeptide chain of the AILIM/ICOS extracellular domain, the target polypeptide chain was divided into four segments, each containing a cysteine residue. Those peptide segments were synthesized by conventional SPPS, followed by thioesterification of the C-terminus. The oligosaccharide moiety, a biantennary complex type disialyloligosaccharide, was attached to the cysteine thiol in the peptide backbone using the haloacetamide method. These peptides, as well as a glycosylated peptide, were sequentially coupled by use of native chemical ligation. This process successfully afforded the desired polypeptide chain having homogeneous oligosaccharides.     

Teratocarcinoma stem cells have a cell surface carbohydrate-binding component implicated in cell-cell adhesion.

Teratocarcinoma stem cells maintained in the undifferentiated state express a carbohydrate-binding component that recognizes oligomannosyl residues. This cell surface molecule is detected by a rosetta assay in which the stem cells form rosettes with glutaraldehyde-fixed trypsinized rabbit erythrocytes. Addition of simple sugars to the assay mixture has little effect, but rosette formation is inhibited by a series of mannose-rich glycoproteins (yeast invertase, yeast mannans and horseradish peroxidase). Periodate oxidation eliminates the inhibitory activity of invertase whereas pronase digestion has little effect, indicating that carbohydrate moieties are essential for inhibition. Invertase and its glycopeptide derivatives also inhibit the reaggregation of dispersed stem cells and promote the dissociation of preformed aggregates. These results suggest that intercellular adhesion of teratocarcinoma stem cels may be the consequence of the interaction of a lectin-like component detected in the rosette assay with a complementary oligosaccharide receptor on adjacent cells.     

Study of the irreversible conformation change of immunoglobulin M by spin-labeling at the carbohydrate and peptide moieties of the molecule

The irreversible conformational change of the immunoglobulin M (IgM) molecule (Waldenstr?m disease) at pH approximately 3 was studied by means of spin-labels introduced in the carbohydrate (2,2,6,6,-tetramethyl-4-aminopiperidine-1-oxyl) and peptide (2,2,5,5,-tetramethyl-3-(dichloro-symm.-triazinylamino)-pyrrolidine-1-oxyl) moieties of the molecule. A marked rise of structure density of IgM especially in the (Fc)5-region and some minor local conformational changes in the Fab-regions were found. Comparison of our findings with the published data shows that Fab-regions of the principal immunoglobulins are rigid structures. Steric hindrance for Fab-regions increases markedly in the row Fab--F(ab')2--IgG--IgA--IgM restricting their spatial mobility. Monomeric Fc-regions of IgM are evidently flexible and one of the domains is especially mobile. It is supposed that oligosaccharide groups of IgM are of two types which differ in their spatial mobility. It was found by ammonium sulfate precipitation of IgM spin-labeled at the peptide moiety that the relative mobility of amino acid residues coupled with spin-label is strongly restricted.     

Artificial peptide and carbohydrate antigens. Immobilization of haptens and adjuvant (MDP) on polyacrylamide

To study the influence of the polyacrylamide carrier on immunogenic properties of the peptide and oligosaccharide haptens, we have prepared artificial antigens by conjugation of a synthetic hexapeptide (homologous to the fragment 95-100 of the murine H-2Db antigen heavy chain) or of an oligosaccharide (antigenic determinant of human blood groops, Lea) with polyacrylamide. In some cases the conjugates containing also a synthetic glycopeptide adjuvant, N-acetylmuramoyl-L-alanyl-D-isoglutamine (MDP), were used. Antisera against haptens were obtained by immunization of BALB/c mice with corresponding conjugates. By the enzyme-linked immunosorbent assay it was shown that these antisera had a high binding titer (up to 10 000) to corresponding hapten, and MDP immobilized on the same carrier as hapten possessed a considerable immunostimulating activity. Thus, usefulness of polyacrylamide for preparation of immunogenic artificial molecules carrying peptide and oligosaccharide haptens was demonstrated.     

A novel glycosulphatase isolated from a mucus glycopeptide-degrading Bacteroides species

Abstract A Bacteroides species which can grow on mucus glycopeptide as energy source, has been used to study sulphate removal from the oligosaccharide chains of mucus glycopeptide. Both cells and extracts removed a portion of the sulphate groups from the mucus glycopeptide. Using sulphate removal from glucose 6-sulphate to assay activity, a novel glycosulphatase located in the periplasmic space was purified 121-fold. The most purified preparations did not remove sulphate from mucus glycopeptide unless another fraction containing glycosidase activity was added back. This result suggests that sulphate is removed after glycosidases have rendered the sulphate group(s) in the mucus glycopeptide oligosaccharide chain accessible.     

Molecular motions of a glycopeptide from human serum transferrin studied by 13C nuclear magnetic resonance.

The molecular motions of a 21-amino-acid glycopeptide (Gp21) containing multiple glycoforms of an N-linked diantennary oligosaccharide were studied by two-dimensional 1H-detected 13C relaxation measurements at natural abundance. Gp21 was derived from human serum transferrin, its amino acid sequence is QQQHLFGSNVTDCSGNFCLFR, and its N-glycan structure is [Formula: see text] The measured longitudinal and transverse relaxation rate constants and the nuclear Overhauser enhancements for the methine carbons of Gp21 were analyzed by using the model-free approach to obtain information about the internal motions in the molecule. The calculated order parameters S2 of the alpha carbons in both NH2- and COOH-terminal segments of the peptide are smaller than those in the interior segment of the Gp21 peptide moiety, implying that the internal motions in the terminal segments are less restricted than in the interior segment. The average S2 value is 0.72-0.91 for the glycosyl residues in the pentasaccharide core of Gp21, 0.58-0.59 for the interior GlcNAc-5,5' residues in the two branches, and 0.35-0.51 for the terminal GlcNAc-5, Gal-6,6', and NeuAcN,N' residues in the two branches, indicating that the internal motions in the glycan core are more restricted than in the two branches.     

The Carbohydrate Moiety of IgM From Atlantic Salmon (Salmo salar L.)

The carbohydrate moiety of salmon IgM was estimated to be about 12.5% of the total molecular weight of salmon IgM based on SDS-PAGE analysis and ≤8.0% based on FACE analysis. The carbohydrate moiety was restricted to the heavy chain and was all N-linked. Six different oligosaccharides were identified using the FACE oligosaccharide profiling technique. Monosaccharide composition analysis, as well as digestion with endoglycosidase H, suggested that the oligosaccharides were mainly of the complex type rather than high mannose type. Removal of about 80% of the carbohydrate affected the sensitivity of IgM to trypsin but had no effects on antigen binding or the complement fixation ability of anti s-RBC IgM.     

Chemo-enzymatic synthesis of a bioactive peptide containing a glutamine-linked oligosaccharide and its characterization

A bioactive peptide containing a glutamine-linked oligosaccharide was chemo-enzymatically synthesized by use of the solid-phase method of peptide synthesis and the transglycosylation activity of endo-β-N-acetylglucosaminidase. Substance P, a neuropeptide, is an undecapeptide containing two l-glutamine residues. A substance P derivative with an N-acetyl-d-glucosamine residue attached to the fifth or sixth l-glutamine residue from the N-terminal region was chemically synthesized. A sialo complex-type oligosaccharide derived from a glycopeptide of hen egg yolk was added to the N-acetyl-d-glucosamine moiety of the substance P derivative using the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis, and a substance P derivative with a sialo complex-type oligosaccharide attached to the l-glutamine residue was synthesized. This glycosylated substance P was biologically active, although the activity was rather low, and stable against peptidase digestion. The oligosaccharide moiety attached to the l-glutamine residue of the peptide was not liberated by peptide-N⁴-(N-acetyl-β-d-glucosaminyl) asparagine amidase F.