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Most cell surface molecules are glycoproteins consisting of linear arrays of globular domains containing stretches of amino acid sequence with similarities to regions in other proteins. These conserved regions form the basis for the classification of proteins into superfamilies. Recombinant soluble forms of six leukocyte antigens belonging to the Ly-6 (CD59), scavenger receptor (CD5), and immunoglobulin (CD2, CD48, CD4, and Thy-1) superfamilies were expressed in the same Chinese hamster ovary cell line, thus providing an opportunity to examine the extent to which N-linked oligosaccharide processing might vary in a superfamily-, domain-, or protein-dependent manner in a given cell. While we found no evidence for superfamily-specific modifications of the glycans, marked differences were seen in the types of oligosaccharides attached to individual proteins within a given superfamily. The relative importance of local protein surface properties versus the overall tertiary structure of the molecules in directing this protein-specific variation was examined in the context of molecular models. These were constructed using the 3D structures of the proteins, glycan data from this study, and an oligosaccharide structural database. The results indicated that both the overall organization of the domains and the local protein structure can have a large bearing on site-specific glycan modification of cells in stasis. This level of control ensures that the surface of a single cell will display a diverse repertoire of glycans and precludes the presentation of multiple copies of a single oligosaccharide on the cell surface. The glycans invariably shield large regions of the protein surfaces although, for the glycoproteins examined here, these did not hinder the known active sites of the molecules. The models also indicated that sugars are likely to play a role in the packing of the native cell surface glycoproteins and to limit nonspecific protein-protein interactions. In addition, glycans located close to the cell membrane are likely to affect crucially the orientation of the glycoproteins to which they are attached.

The extracellular domain of E2 glycoprotein outer surface of the classical swine fever virus was expressed in epithelial kidney pig cells. The N-glycosylation determined by combination of Normal Phase-HPLC, Weak Anion Exchange-HPLC, exoglycosidase digestions and Mass Spectrometry revealed a complex mixture of neutral and monosialylated multiantennary N-glycans with variable number of alpha1-3-Gal-Gal antennae terminals. The most abundant neutral N-glycan has a composition of Hex(7)HexNAc(4)dHex(1), Negative ion ESI-MS/MS confirmed the presence of the alpha1-3-Gal-Gal motif on each arm of the fucosylated biantennary N-glycan. The most abundant monosialylated glycan was Hex(6)HexNAc(4)dHex(1)Neu(5)Ac(1), with the sialic acid linked to the terminal beta1-4-Gal-GlcNAc. Sialic acid on the antenna capping position was predominantly of the N-acetyl form.

Endolyn (CD164) is a sialomucin that functions as an adhesion molecule and a negative regulator of CD34+ CD38- human haematopoietic precursor cell proliferation. The 105A5 and 103B2/9E10 CD164 monoclonal antibodies (mAbs), which act as surrogate ligands, recognize distinct glycosylation-dependent classes I and II epitopes located on domain I of the native and recombinant CD164 proteins. Here, we document five new CD164 mAbs, the 96 series, that rely on conformational integrity, but not glycosylation, of exons 2- and 3-encoded CD164 domains, thereby resembling the class III mAbs, N6B6 and 67D2. Although all the 96 series class III mAbs labelled both the 105A5+ and 103B2/9E10+ cells, cross-competition and immunoblotting studies allow them to be categorized into two distinct class III subgroups, i.e. the N6B6-like subgroup that only recognizes 80-100 kDa proteins and the 67D2-like subgroup that also recognizes a higher molecular weight (>220 kDa) form. To more closely define the reactivity patterns of mAbs to the classes I and II epitopes, the global glycosylation patterns of the soluble human (h) CD164 proteins were determined using lectin binding, high-performance liquid chromatography (HPLC) and mass spectrometry. hCD164 recombinant proteins bound to the lectins, Galanthus nivalis agglutinin, Datura stramonium agglutinin, Sambucus nigra agglutinin, Maackia amurensis agglutinin and peanut agglutinin, indicating the presence of high mannose and complex N-glycans, in addition to core 1 O-glycans (the Tn antigen) and alpha2-3 and alpha2-6 sialic acid moieties. Our HPLC and mass spectrometry results revealed both high mannose and complex N-glycosylation with various numbers of branches increasing the complexity of the glycosylation pattern. Most O-glycans were small, core 1 or 2 based. High levels of sialylation in alpha2-3 and alpha2-6 linkages, without sialyl-Lewis X, indicate that the majority of these hCD164 recombinant proteins are unable to bind to selectins in our assay system, but may interact with Siglec molecules.

The combination of experimental and theoretical techniques gives considerable insight into oligosaccharide conformation. Through this, it became relatively straightforward to characterize the conformational properties of individual glycosidic linkages. Different types of linkages show different types of conformational behavior, one or more conformers, and different degrees of flexibility. A single type of linkage can show different behavior in different environments, the conformational freedom being further reduced by the surrounding residues.

The relatively large size and dynamics of oligosaccharides can result in substantial shielding of functionally important areas of proteins to which they are attached, modulate the interactions of glycoconjugates with other molecules and affect the rate of processes which involve conformational changes. This review focuses on the occupancy of N-linked glycosylation sites on three enzymes, ribonuclease, plasminogen and tissue plasminogen activator. Each of these proteins occurs naturally as two populations of molecules, distinguished from each other only by the presence or absence of an oligosaccharide at one glycosylation site. The presence of an oligomannose sugar on ribonuclease (at Asn-34) alters its overall dynamics, increases its stability towards proteinases and decreases its functional activity towards double-stranded RNA. The N-linked sugar on plasminogen (at Asn-288) within kringle 3 reduces the rate of the beta- to alpha-conformational change, modulates the transport of plasminogen into the extravascular compartment, decreases plasminogen binding to U937 cells and downregulates the activation of plasminogen by both urokinase and tissue plasminogen activator. Additionally, in fibrinolysis, within a ternary complex of fibrin, plasminogen and tissue plasminogen activator, the N-linked sugar of plasminogen hinders the initial interaction with tissue plasminogen activator (i.e., it alters Km). The presence of an N-linked glycan (at Asn-184) in the kringle 2 domain of tissue plasminogen activator hinders the rearrangement of this ternary complex, decreasing the turnover rate (Kcat).

The Ca(2+)-dependent carbohydrate-recognition domain (CRD) of rat serum mannose-binding protein has been subjected to site-directed mutagenesis to determine the importance of individual residues in ligation of mannose and related sugars. The effects of the mutations were assessed by direct binding assays, competition binding studies, partial proteolysis, and NMR analysis of sugar-CRD titrations. As suggested by the crystal structure of the mannose-binding CRD complexed with oligosaccharide ligand, asparagine and glutamic acid residues that interact with hydroxyl groups 3 and 4 of the sugar, as well as with one of the two bound Ca2+, are critical for ligand binding. In addition, the beta-carbon of His189 contributes substantially to the binding affinity, apparently through a van der Waals contact with C-4 of the sugar ligand. van der Waals contacts between the imidazole ring of His189 and the 2 hydroxyl group of mannose, and between Ile207 and C-6 of mannose, observed in the crystal structure, contribute less to stability of the ligand complex. The effects of changes at positions 189 and 207 on the ability of the CRD to distinguish between alpha-and beta-methyl L-fucosides suggest that fucose may bind in an alternative orientation compared to the arrangement originally proposed based on the mannose-CRD complex.

Hydrophobic vitamins are transported in human plasma and extravascular fluids by carrier proteins. No specific protein has been described so far for vitamin E, which plays a crucial role in protecting against oxidative damage and disease. We report here the purification of a 75-kDa glycoprotein with vitamin E-binding properties by stepwise chromatography of lipoprotein-depleted human plasma and monitoring of vitamin E (alpha-tocopherol)-binding activity. Partial sequencing identified this protein as afamin, a previously described member of the albumin gene family with four or five potential N-glycosylation sites. Glycosylation analysis indicated that >90% of the glycans were sialylated biantennary complex structures. The vitamin E-binding properties were confirmed using recombinantly expressed afamin. Qualitative and quantitative analysis of plasma and extravascular fluids revealed an abundant presence of this protein not only in plasma (59.8+/-13.3 microg/mL) but also in extravascular fluids such as follicular (34.4+/-12.7 microg/mL) and cerebrospinal (0.28+/-0.16 microg/mL) fluids, suggesting potential roles for afamin in fertility and neuroprotection. Afamin is partly (13%) bound to plasma lipoproteins. Afamin and vitamin E concentrations significantly correlate in follicular and cerebrospinal fluids but not in plasma. The vitamin E association of afamin in follicular fluid was directly demonstrated by gel filtration chromatography and immunoprecipitation which complements the in vitro findings for purified native and recombinant afamin.

The human complement regulator CD55 is a key molecule protecting self-cells from complement-mediated lysis. X-ray diffraction and analytical ultracentrifugation data reveal a rod-like arrangement of four short consensus repeat (SCR) domains in both the crystal and solution. The stalk linking the four SCR domains to the glycosylphosphatidylinositol anchor is extended by the addition of 11 highly charged O-glycans and positions the domains an estimated 177 Å above the membrane. Mutation mapping and hydrophobic potential analysis suggest that the interaction with the convertase, and thus complement regulation, depends on the burial of a hydrophobic patch centered on the linker between SCR domains 2 and 3.

Proteins regulate wound healing. Knowledge of the proteins present in poorly healing wounds may reveal new targets for treatment. This article describes how proteomics can be used to help identify the proteins involved in wound healing.

A family of about 20 novel acidic bi- and tri-antennary N-glycans, amounting to almost half those expressed on Bowes melanoma tissue-plasminogen activator (t-PA) were found to possess Galbeta1--4GlcNAcbeta1--, sulfated and sialylated GalNAcbeta1--4GlcNAcbeta1--or sulfated GlcAbeta1--3Galbeta1--4GlcNAcbeta1--antennae, of which those containing sulfated GlcA, depicting the L2/HNK-1 carbohydrate epitope, were preferentially located on the 6 arm. A proportion of the glycans were highly charged, because of multiple and variously distributed sulfation, some of which was located on the fucosylated chitobiose core. Multiple expression of the L2/HNK-1 epitope on a single glycan was observed. The most abundant compound was a biantennary glycan carrying sulfated GlcA on the 6-branched antenna and an alpha2--6 sialylated GalNAc on the other. The N-glycosylation sequon containing Asn448, which is known to express all of the sulfate-carrying N-glycans contains, unusually, an arginine residue. An electrostatic interaction between this cationic amino acid and the core-sulfate group of the N-glycan is proposed to reduce mobility of the carbohydrate in the region of the t-PA active site. Because of the 'brain-type' nature of the N-glycans described in this neuro-ectodermal cell line, the possibility of neural t-PA interacting with the L2/HNK-1-recognizing molecule, laminin, of the central nervous system extracellular matrix is discussed.

Genomics establishes the relationship between biological processes and gene activity. Proteomics (James 1997), which relates biological activity to the proteins expressed by genes, is fundamental to our understanding of biology. It is the proteins, rather than the genes that encode them, which engage in biological events (Wilkins et al. 1995). Furthermore, most proteins contain post-translational modifications which are the products of enzyme reactions. Since the enzymes are coded for by different genes, the complete structure of an individual protein cannot be determined by reference to either a single gene or the protein sequence alone. One of the most common ways that a protein is modified is by the process of glycosylation, in which oligosaccharides are attached to specific sites encoded in the primary sequence of the protein (Dwek 1996).

The organ-specific nature of the glycosylation of Na+,K+-ATPase-enriched preparations from kidney and brain tissues has earlier been indicated by the use of lectin-staining techniques. Na+,K+-ATPase is ubiquitous and abundant, and subject to upregulation during cell-division and in certain pathological conditions. Lectins specific for the different carbohydrates displayed by the Na+,K+-ATPases may, therefore, be useful carriers/mediators in tissue-specific targeting. N-linked oligosaccharides purified from Na+,K+-ATPase-enriched preparations from rat brain and kidney were consequently characterised in detail in this study using weak anion exchange and normal phase HPLC (combined with serial glycosidase digestions) and matrix-assisted laser desorption/ionisation mass spectrometry. The oligomannose series of glycans were most abundant in the brain tissue preparation and this contrasted with the renal-associated oligosaccharides that were dominated by families of tetra-antennary glycans (with/without a core fucose) with up to four lactosaminylglycan residues in either branched or linear formation.

Melanin biosynthesis is completely inhibited in the B16 melanoma cells following their incubation with inhibitors of the two ER glucosidases. This is primarily due to the inactivation of tyrosinase. Under the same conditions, the DOPA-oxidase activity of TRP-1 was only partially affected. In this report we investigate the effects of the perturbation of N-glycan processing in ER on the transport and activation of tyrosinase and TRP-1. We have localized the DOPA-oxidase activity in normal and inhibited cells and suggest that the first DOPA-reactive compartment of the secretory pathway (trans Golgi network) is also the site of tyrosinase activation. The inhibition of N-glycan processing does not affect the intracellular trafficking of the two melanogenic enzymes that are correctly transported to melanosomes. Immunoprecipitation experiments followed by analysis in SDS-PAGE under non-reducing conditions suggest that in inhibited cells, both tyrosinase and TRP-1 are synthesized in a modified conformation as compared to the normal proteins. These data suggest that the inhibition of melanin synthesis is not due to a defective transport but rather to conformational changes induced in the structure of tyrosinase and TRP-1 during their transit through the ER.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of N-linked carbohydrates (those linked to asparagine in glycoproteins) was first reported in 1991 by Mock et al. [1] and provides a simple and sensitive method for producing spectra from underivatized N-linked glycans. It has been estimated that MALDI is about 10 to 100 times more sensitive than fast-atom bombardment FAB) for glycan analysis [2], and does not require derivatization by permethylation or peracetylation, as required by FAB, to obtain a signal. When recorded with linear time-of-flight (TOF) mass spectrometers, MALDI mass spectra of neutral N-linked glycans contain few, if any, fragment ions and so the technique provides a rapid method for glycan profiling. The spectrum of N-linked glycans released from chicken ovalbumin, shown in Fig. 1 illustrates the type of result that can be obtained. Structures of the glycans producing the peaks are shown in Table 1. The masses of the peaks can lead directly to the compositions ofthe constituent glycans in terms oftheir isobaric monosaccharide compositions, but determination of further structural details requires production of fragment ions or coupling to other methods such as exoglycosidase digestion as described below. Acidic N-linked glycans produce more fragmentation than neutral glycans and are generally more difficult to handle. Nevertheless, they provide excellent spectra under the right conditions. This paper summarizes work in this area and presents examples of data recorded in our laboratory.

Tyrosinase and tyrosinase-related protein-1 (TRP-1) are two melanogenic enzymes that regulate melanin biosynthesis. Both are glycoproteins and belong to the TRP-1 gene family. They share a significant level of sequence similarity in several regions, including the catalytic domain and the potential N-glycosylation sites. We have recently shown that inhibition of the early steps of N-glycan processing in B16F1 cells dramatically affects tyrosinase activity and melanin synthesis. We present here results on N-glycan processing of TRP-1 and tyrosinase and compare the maturation process and activity of both glycoproteins in the presence of inhibitors of the endoplasmic reticulum stages of N-glycosylation. N-glycan analysis reveals that each of these two glycoproteins contains a mixture of high-mannose and sialylated complex N-glycans. However, in contrast to TRP-1, tyrosinase presents a homogeneous high-mannose glycoform, also. In the presence of alpha-glucosidases inhibitors, the maturation of tyrosinase N-glycans is completely inhibited, whereas TRP-1 is still able to acquire some complex glycans, indicating that endomannosidase acts preferentially on the later glycoprotein. In addition, the dopa-oxidase activity of tyrosinase is totally abolished, whereas for TRP-1 it is only partially affected. The results suggest that despite their structural similarity, tyrosinase is more sensitive than TRP-1 to perturbations of early N-glycan processing, in terms of maturation and catalytical activity.

Glycosylation is the primary cause of microheterogeneity in proteins (glycoforms). These reflect complexity at both molecular and cellular levels. Protein sugar prints are conserved and not random under normal physiological conditions. There are many potential functions of glycosylation. For instance, physical properties include: folding, trafficking, packing, stabilisation, protease protection, quaternary structure and organization of water structure. Properties relating to recognition and biological triggering are characterized by: weak interactions, multiple presentation and precise geometry. Many of the properties may only operate in a specific biological context. Changes in sugar prints may both reflect and results in physiological changes, e.g. cancer and rheumatoid arthritis. It is necessary, in many systems, to evaluate glycosylation. This requires accurate monitoring of sugar prints of using automated and predictive technology. Glycosylation often affords a sensitive means of monitoring pharmaceutical products for Quality Control.

We have characterized the glycans at individual sites on the hemagglutinin of three influenza A variants to obtain information on the role of cell-specific glycosylation in determining the receptor binding properties of this virus. The variants differ in whether they have a glycosylation site at residue 129 on the tip of the hemagglutinin and whether amino acid 184 (near to the receptor binding site) is His or Asn. We found that all sites on each variant are glycosylated in Madin-Darby bovine kidney cells, that the glycosylation is site-specific, and that the glycans at the same site in each variant are highly similar. One site that is buried in the hemagglutinin trimer contains only oligomannose glycans. The remaining sites carry complex glycans of increasing size as the distance of the site from the viral membrane decreases. Most of these complex glycans are terminated with alpha-galactose residues, a consequence in bovine cells of the removal of terminal sialic acids by the viral neuraminidase. Although the glycans at residue 129 are among the smallest on the molecule, they are large enough to reach the receptor binding pocket on their own and adjacent monomers. The results suggest that the reduction in receptor binding observed with Madin-Darby bovine kidney cell-grown virus is due to the combined effect of large complex glycans at the tip of the hemagglutinin and a His to Asn substitution close to the receptor binding pocket.

The sugars released from a pure glycoprotein often consist of a heterogeneous population containing both neutral and charged oligosaccharides. For example, the single N-glycosylation site in human erythrocyte CD59 is associated with more than 100 neutral and sialylated complex glycans, each representing a different glycoform (1). The existence of such extensive heterogeneity in biologically important glycoproteins requires refined approaches to the analysis of oligosaccharides. The adaptable technology which is described here represents a significant advance towards faster, more automated and more detailed strategies for the rapid profiling and analysis of sugars. Such technologies may be required for major studies, such as the human genome project, which defines DNA in normal and diseased states, and the proteome project, which sets out to analyse the total amount of protein in a living cell. It is worthy of note that genetic diseases are not caused by the genes themselves, but by the products for which the genes code and their post-translational modifications, which include glycosylation. In this chapter two strategies for rapid oligosaccharide analysis are described: Oligosaccharide profiling and detailed structural analysis.

Immunoglobulin G (IgG) is glycosylated in both the Fc and the Fab regions of the protein with a heterogeneous ensemble of structures (glycoforms) that is both highly reproducible (i.e. nonrandom) and site specific. In normal IgG, the 2 highly conserved oligosaccharides of the Fc region are found buried between the CH2 domains, forming specific protein-saccharide interactions with the Fc protein surface. One of the functions attributed to the Fc oligosaccharides of normal IgG is to maintain the conformational arrangements of the Fc domains as well as the hinge regions. These structural features are necessary for Fc effector functions such as Clq and monocyte binding. A hallmark of rheumatoid arthritis (RA) patients is a dramatic increase in the presence of serum IgG containing Fc oligosaccharides lacking an outer arm galactose residue (termed 'G0' glycoforms). The increased level of G0 has been shown to be directly related to the pathogenesis of RA. Nuclear magnetic resonance relaxation studies of the Fc region from normal and RA IgG, as well as examination of x-ray structures, show that the G0 oligosaccharides have an increased mobility resulting from the loss of binding between the G0 oligosaccharide and the Fc protein surface. From these observations it follows that regions of the protein surface that are normally covered by the oligosaccharide are revealed. The newly accessible protein surface could have lectin-like activity and also be inherently antigenic. In addition, the more mobile G0 oligosaccharide can be recognised by mannose binding protein. As the mannose binding protein can activate complement, and the Fc oligosaccharide would not normally be accessible to protein recognition, this finding might suggest a specific role for the G0 glycoform in inflammation when the appropriate IgG glycoforms are clustered.

A receptor which binds secretory IgA (sIgA) is expressed on human T cells from patients with systemic lupus erythematosus, rheumatoid arthritis, Behcet's syndrome and IgA nephropathy and on normal T cells following phytohaemagglutinin (PHA) stimulation. The specificity of this receptor was initially probed with a panel of normal serum immunoglobulins in competitive inhibition assays with sIgA using two-colour immunofluorescence. While the receptor showed the strongest affinity for IgA1 (IC(50)10(-6) M), IgD which has a similarly glycosylated hinge region to IgA1, also bound to the receptor (IC50 10(-5) M). IgA2, which lacks the 'O'-glycosylated hinge region, did not significantly inhibit the binding at these concentrations suggesting that the IgA determinants for this receptor might be the oligosaccharides present in the hinge region of IgA1. IgA1 has up to 10 'O'-linked oligosaccharides and four N-linked oligosaccharides per molecule. In order to probe the role of the 'O'-linked hinge sugars in the binding event, a sugar library was prepared from IgA1 by a procedure designed to release 'O'-linked oligosaccharides preferentially, and to retain them in the natural closed ring formation. The sugars were released by hydrazinolysis at 65 degrees and the resulting oligosaccharide library analysed by high voltage paper electrophoresis (HVE) and P4 gel permeation chromatography. Competitive inhibition studies demonstrated that both the library and the individual 'O'-linked sugars associated with IgA1 were implicated in the binding of IgA1 to this receptor (IC50 between 1 x 10(-5) M and 6 x 10(-5) M). Within this range the individual sugars showed small differences in their affinity for the receptor in the following order: Gal beta 3GalNAc = NeuNAc2 alpha 3(6)Gal beta 3GalNAc > NeuNAc2 alpha 3(6)Gal beta 3[NeuNAc2 alpha 6]GalNAc > or = GalNAc.

Cytotoxic T cells (CTL) recognize short peptide epitopes presented by class I glycoproteins encoded by the major histocompatibility complex (MHC). It is not yet known whether peptides containing posttranslationally modified amino acids can also be recognized by CTL. To address this issue, we have studied the immunogenicity and recognition of a glycopeptide carrying an O-linked N-acetylglucosamine (GlcNAc) monosaccharide-substituted serine residue. This posttranslational modification is catalyzed by a recently described cytosolic glycosyltransferase. We show that glycosylation does not affect peptide binding to MHC class I and that glycopeptides can elicit a strong CTL response that is glycopeptide specific. Furthermore, glycopeptide recognition by cytotoxic T cells is dependent on the chemical structure of the glycan as well as its position within the peptide.

The rat and human forms of the T-cell surface glycoprotein CD4 share a common glycosylation site at the Asn270/271 position but differ with respect to the locations of the second glycosylation sites at Asn159 (rat) and Asn300 (human). The glycosylation of soluble recombinant forms of human and rat CD4 (sCD4) expressed in Chinese hamster ovary cells has been characterized. The most obvious differences between the rat and human sCD4 oligosaccharides were the greater abundance of oligomannose and hybrid oligosaccharides on rat sCD4 and the presence of oligosaccharides carrying a terminal alpha-galactose residue on human sCD4. This is the first report of the occurrence of alpha-galactose residues on a glycoprotein expressed in Chinese hamster ovary cells. Comparison of mutant rat sCD4 molecules with single glycosylation sites and glycopeptides indicated that site-specific and independent processing occurred at each glycosylation site. The glycosylation at the conserved site at Asn270 of rat sCD4 was identical to that seen for the equivalent site in human sCD4, and the oligomannose and hybrid structures were restricted to the nonconserved site at Asn159 in rat sCD4. However, there was more oligosaccharide processing at this site in a truncated form of rat sCD4 consisting of the two NH2-terminal domains. These results indicate that not only the local three-dimensional structure but also domain interactions can influence the processing at individual glycosylation sites.

The genetic selection of mutant cells in a glycoconjugate biosynthetic pathway may provide insights into metabolic disease.

A method of oligosaccharide analysis involving controlled fragmentation resulting from enzymatic digestion is presented. The principle involves generating a set of fragments from the original oligosaccharides, characterizing them in terms of their hydrodynamic volumes, determining their molar proportions, and identifying the oligosaccharides by comparison with a computer-generated data base. Experimentally, this technique involves incubation of aliquots of a sample with a set of defined mixtures of exoglycosidases followed by pooling of the products and a single analysis on the product pool. This method has several practical advantages over current techniques, including speed and the ability to use smaller amounts of starting material. The detection of the intensity-versus-hydrodynamic volume profile is limited only by the specific activity of the labeling method. The ability to perform the enzyme digestions is limited by the individual Km values of the enzymes.

The complete primary structures of the major Asn-linked oligosaccharides from the type II variant surface glycoproteins (VSGs), MITat 1.2 and MITat 1.7, and the type III VSG, MITat 1.5, were determined using a combination of exo- and endoglycosidase digestions, methylation analysis, acetolysis, and 500 MHz 1H NMR spectroscopy. Each variant contained classical branched oligomannose-type and biantennary complex oligosaccharides, a proportion of the latter substituted with terminal alpha(1-3)-linked galactose residues, the first report of the presence of this epitope in Trypanosoma brucei. In addition both the type II variants contained relatively large amounts of the unusual small oligomannose-type oligosaccharides, Man4GlcNAc2 and Man3GlcNAc2, and a diverse array of novel branched poly-N-acetyllactosamine oligosaccharides, similar but not identical to those from mammalian glycoproteins. These latter structures were also partially substituted with terminal alpha(1-3)-linked galactose residues. Glycosylation in the type II variants showed site specificity in that the poly-N-acetyllactosamine and Man(9-5)GlcNAc2 oligosaccharides were located exclusively at Asn-glycosylation site 1 very close to the C terminus, whereas the Man(4-3)GlcNAc2 and biantennary complex oligosaccharides were located exclusively at site 2. This is the first report of the presence of poly-N-acetyllactosamine oligosaccharides in protozoa.

SAP isolated in pure form from pooled normal serum (5000 individual donors), from pooled malignant effusion fluids (500 individual donors) and from single individuals, including one with AA amyloidosis, was characterised with respect to the following properties: 1) its binding activity for the synthetic ligand, phosphoethanolamine-Sepharose and for AL amyloid fibrils; 2) its behaviour on reverse phase chromatography; 3) its isoelectric focussing pattern in urea Polyacrylamide gels; 4) its metabolism in normal mice and mice with AA amyloidosis [1]; 5) its complete glycan structure. Remarkably, no heterogeneity or polymorphism was observed. Even the glycan, of which there is a single N-linked biantennary chain per SAP subunit, displays none of the microheterogeneity characteristic of almost all other known glycoproteins. SAP must thus have important function (s) strictly related to its structure. Carbohydrate analysis of AP samples from 3 different individuals predominantly showed a structure identical to SAP but another glycan with terminal N-acetyl glucosamine residues was also present at 3-4% and was not found in normal SAP or SAP from non-amyloid patients.

The C1 inhibitor component of human complement is a member of the serpin superfamily, and controls C1 activation. Carbohydrate analyses showed that there are seven O-linked oligosaccharides in C1 inhibitor. Together with six N-linked complex-type oligosaccharides, the carbohydrate content is therefore 26% by weight and the molecular weight (Mr) is calculated as 71,100. Neutron scattering gives an Mr of 76,000 (+/- 4000) and a matchpoint of 41.8 to 42.3% 2H2O, in agreement with this carbohydrate and amino acid composition. Guinier plots to determine the radius of gyration RG were biphasic. Neutron contrast variation of C1 inhibitor in H2O-2H2O mixtures gave an overall radius of gyration RG at infinite contrast of 4.85 nm, from analyses at low Q, and a cross-sectional RG of 1.43 nm. The reactive centre cleaved form of C1 inhibitor has the same Mr and structure as the native molecule. The length of C1 inhibitor, 16 to 19 nm, is far greater than that of the putative serpin domain. This is attributed to an elongated structure for the carbohydrate-rich 113-residue N-terminal domain. The radial inhomogeneity of scattering density, alpha, is large at 59 x 10(-5) from the RG data and 28 x 10(-5) from the cross-sectional analysis, and this is accounted for by the high oligosaccharide content of C1 inhibitor. The scattering data were modelled using small spheres. A two-domain structure of length 18 nm based on two distinct scattering densities accounted for all the contrast variation data. One domain is based on the crystal structure of alpha 1 antitrypsin (7 nm x 3 nm x 3 nm). The other corresponds to an extended heavily glycosylated N-terminal domain of length 15 nm, whose long axis is close to the longest axis of the serpin domain. Calculation of the sedimentation coefficient s0(20),w for C1 inhibitor using the hydrodynamic sphere approach showed that a two-domain head-and-tail structure with an Mr of 71,000 and longest axis of 16 to 19 nm successfully reproduced the s0(20),w of 3.7 S. Possible roles of the N-terminal domain in the function of C1 inhibitor are discussed.

The extent of protein N-glycosylation in Leishmania mexicana amazonensis has been proposed to be a factor in the virulence of the parasite. The N-linked oligosaccharides of gp63, the major surface glycoprotein of L. mexicana amazonensis, were characterized after their release by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. High voltage paper electrophoresis of the reduced oligosaccharides revealed only neutral species. Gel-permeation chromatography on Bio-Gel P-4 yielded four fractions, and the oligosaccharides present were structurally characterized by sequential exoglycosidase digestion, fragmentation by acetolysis, and methylation analysis. Four major structures were found and were biantennary oligomannose type with compositions of Glc1Man6GlcNAc2 (La), Man6GlcNAc2 (Lb), Man5GlcNAc2 (Lc), and Man4GlcNAc2 (Ld). The largest oligosaccharide (La) was shown to contain a terminal glucopyranosyl residue on the alpha (1----3) arm. The biantennary oligomannose structures (Lb and Lc) and the glucosylated structure Glc1Man6GlcNAc2 (La) have not previously been reported as a component of a mature glycoprotein from any source.

In rheumatoid arthritis an increased proportion of the N-linked oligosaccharides on serum IgG terminate with N-acetylglucosamine (agalactosyl IgG). It has recently been shown that group A streptococcal cell wall peptidoglycan/polysaccharide complex may be used to raise monoclonal antibodies which bind to this glycoform of IgG. Patients with rheumatoid arthritis also have increased levels of antibody to the 65 kD and 70 kD families of heat shock proteins, particularly to a bacterial (Mycobacterium bovis) homologue of heat shock protein hsp65. Streptococci must contain similar heat shock proteins. Acute rheumatic fever follows infection with group A streptococci, and these organisms might theoretically evoke antibody to heat shock proteins or changes in the levels of agalactosyl IgG, which is antigenically cross reactive with their cell walls. It is shown here that serum samples from patients with acute rheumatic fever do not differ from those from normal children by these criteria.

A number of eukaryotic surface glycoproteins, including the variant surface glycoproteins of Trypanosoma brucei, are synthesized with a carboxyl-terminal hydrophobic peptide extension that is cleaved and replaced by a complex glycosyl-phosphatidylinositol (GPI) membrane anchor within 1-5 min of the completion of polypeptide synthesis. The rapidity of this carboxyl-terminal modification suggests the existence of a prefabricated precursor glycolipid that can be transferred en bloc to the polypeptide. We have reported the purification and partial characterization of a candidate precursor glycolipid (P2) and of a compositionally similar glycolipid (P3) from T. brucei (Menon, A. K., Mayor, S., Ferguson, M. A. J., Duszenko, M., and Cross, G. A. M. (1988) J. Biol. Chem. 263, 1970-1977). The primary structure of the glycan portions of P2 and P3 have now been analyzed by a combination of selective chemical fragmentation and enzymatic glycan sequencing at the subnanomolar level. The glycans were generated by deamination, NaB3H4 reduction, and dephosphorylation of glycolipids purified from different trypanosome variants. Glycan fragments derived from biosynthetically labeled glycolipids were also analyzed. The cumulative data strongly suggest that P2 and P3 contain ethanolamine-phosphate-Man alpha 1-2Man alpha 1-6Man alpha 1-GlcN linked glycosidically to an inositol residue, as do all the GPI anchors that have been structurally characterized. The structural similarities suggest that GPI membrane anchors are derived from common precursor glycolipids that become variably modified during or after addition to newly synthesized proteins.

On p. 531 of the June 2001 issue, an incorrect version of a figure was printed to accompany the News & Views article entitled “Holistic approaches to glycobiology” by Pauline M. Rudd, Ghislain Opdenakker, and Raymond A. Dwek. The correct version of the figure can be viewed at http://www.nature.com/nbt/journal/ v19/n6/fig_tab/nbt0601_531_F1.html

To probe the effects of N-glycosylation on the fibrin-dependent plasminogenolytic activity of tissue-type plasminogen activator (t-PA), we have expressed a human recombinant t-PA (rt-PA) gene in Chinese hamster ovary (CHO) cells and in a murine C127 cell line. The resulting rt-PA glycoproteins were isolated and their associated N-linked oligosaccharide structures determined by using a combination of high-resolution Bio-Gel P-4 gel filtration chromatography, sequential exoglycosidase digestion, and methylation analysis. The results show that CHO rt-PA is N-glycosylated differently from murine C127 derived rt-PA. Further, both rt-PA's are N-glycosylated differently from t-PA derived from a human colon fibroblast and the Bowes melanoma cell line (Parekh et al., 1989), confirming that N-glycosylation of the human t-PA polypeptide is cell-type-specific. Both CHO and murine rt-PA were fractionated on lysine-Sepharose chromatography. The N-glycosylation of the major forms was analyzed and their fibrin-dependent plasminogenolytic activity determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. The results suggest that the various forms of rt-PA differ from one another with respect to the kinetics of their fibrin-dependent activation of plasminogen. Together, these data support the notion (Wittwer et al., 1989) that N-glycosylation influences the fibrin-dependent catalytic activity of t-PA and that t-PA when expressed in different cell lines may consist of kinetically and structurally distinct glycoforms.

Pre-resonance Raman spectroscopy was used to study the interactions of the nitro groups of dinitrophenyl haptens with three dinitrophenyl-binding antibody fragments: M315 Fv, M460 Fab‘ and X25 Fab’. The observed changes in frequency of modes associated with the nitro moieties are compared with solvent-induced changes for the model hapten 2,4-dinitroaniline. These comparisons demonstrate a specific interaction via the H2N-C-C-2-NO2 and 4-NO2 groups with the protein. The interaction with the 4-NO2 group appears to be absent for epsilon-N-2,4-dinitrophenyl-L-lysine bound to M315 Fv fragment in contrast with either 2,4-dinitrophenylaspartate or 2,4-dinitrophenylglycine bound to M315 Fv fragment, despite the much tighter binding of the lysine derivative. The implications of this for M315 Fv fragment in terms of the antibody specificity are discussed. Comparisons of the effect of binding to M460 Fab‘ and X25 Fab’ fragments also revealed significant differences in the shifts of the nitro group vibrations of 2,4-dinitrophenyl-lysine and 2,4-dinitroaniline.

With the aim of clarifying the relationship between the activation of the first component of complement (C1) by immunoglobulin and by polyanions, the mode of interaction of C1q with DNA was investigated by structural and inhibition studies. DNA inhibits C1q binding to IgG immune complexes (ICs) through binding to C1q rather than to IgG, as seen from two lines of evidence. Firstly, DNA does not bind to ICs under conditions where full binding to C1q is observed. Secondly, at I - 0.15, DNA inhibits more strongly when mixed first with C1q rather than with ICs. The inhibition of C1q-IgG binding by DNA is subject to kinetic factors. Firstly, DNA is not an effective inhibitor if added after C1q has bound to ICs. This at least in part reflects a portion of the IgG-bound C1q that exchanges only very slowly with free C1q. Secondly, the relative rates of association of C1q to DNA and ICs at different ionic strengths are important in determining whether inhibition is observed. The existence of a kinetic effect in the inhibition by DNA means that inhibition experiments cannot be used to establish whether DNa binds to the same site on C1q as IgG. This question was therefore approached by structural studies. Precipitation of C1q with DNA was greatly diminished by heat or pH 4.45 denaturation of C1q, by pepsin digestion to remove the globular heads, and by limited modification with 1, 2-cyclohexanedione. In contrast, extensive modification with methyl acetimidate only had a limited effect. In these respects the structural requirements for C1q-DNA precipitation were similar to those for C1q-Igg binding, as would be consistent with binding of DNA and IgG to nearby or overlapping sites on C1q. In view of residual DNA-precipitating activity in the pepsin fragment preparation of C1q, there is the possibility that there are additional DNA sites on the collagenous tails.

Data from ¹H nuclear magnetic resonance studies on the Fv fragment of protein 315, a Dnp-binding BALB/c mouse lgA(λ2) myeloma protein, have been used to refine a predicted structure of the combining site of the protein. The Dnp-binding subsite in the modified structure is composed of the side chains of three aromatic amino acids Trp 93L, Tyr 34L and 34H. A fourth aromatic amino acid residue is close to the side chain-NH-CH 2 -group, this is Tyr 33 H - The antibody-hapten binding is a simple encounter process, which causes no extensive conformation change in the Fv fragment. A method for paramagnetic structural studies has been devised using Dnp derivatives with cllgophosphate side chains, which create a specific manganese binding site on the Fv fragment-hapten complex. The distances from the bound metal ion to the imidazole side chains of two of the three hlstldine residues of protein 315 have been determined. ¹H nuclear magnetic resonance has been used to study the histidine residues of the Fv fragment of protein 315. It has been shown that one of the three histidine residues (102H) is close to the combining site, but that this residue does not participate directly in binding haptens. 31 P nuclear magnetic resonance studies have shown the presence of a positively charged amino acid side chain near the entrance of the combining site of the Fv fragment. This residue has been identified as Arg 95L. The mode of binding of trini trophenyl derivatives to the Fv fragment has been studied by 1H nuclear magnetic resonance. It is concluded that these haptens, when bound to the Kv fragment, make contacts with the same amino acid side chains as Dnp derivatives.

A phosphorylated 3-deoxy-manno-octulosonic acid (KDO) was released from the lipopolysaccharide (LPS) of the deep rough mutant (Rb+169) of Haemophilus influenzae by acid hydrolysis. Both phosphorylated and dephosphorylated KDO, produced by treatment with alkaline phosphatase, were identified by gas chromatography-mass spectrometry after trimethylsilylation. This technique provides a rapid and reliable method for the identification of phosphorylated KDO in LPS.

It has been proposed that protein-bound oligosaccharides interact with the protein to which they are attached to up- or down-regulate the bioactivity of the 'composite' glycoprotein. Oligosaccharide analyses of the glycoproteins Thy-1, tissue plasminogen activator and immunoglobulin G are presented. Correlations between particular glycoforms and enzymic activities are demonstrated for tissue plasminogen activator. The change in the prevalence of particular immunoglobulin G glycoforms is shown to correlate with disease activity in rheumatoid activity.