A high-performance liquid chromatography based strategy for rapid, sensitive sequencing of N-linked oligosaccharide modifications to proteins in sodium dodecyl sulphate polyacrylamide electrophoresis gel bands.

The majority of biologically active proteins are glycosylated, therefore any approach to proteomics which fails to address the analysis of oligosaccharides is necessarily incomplete. To appreciate the structure of a glycoprotein fully, to understand the roles for the attached oligosaccharides and to monitor disease associated changes it is necessary to visualise the sugars as well as the protein. To achieve this aim when biological samples are available at the low microgram level or less has involved increasing the sensitivity of the technology for glycan analysis. Since one protein may have many different oligosaccharides attached to it (glycoforms) this is a major technical challenge. CD59, for example, has over 100 different sugars at one N-linked glycosylation site. Applications of recently developed technology suggest that it is now becoming realistic to extend the proteomics analysis of glycoproteins to include details of glycosylation. This is achieved by releasing the N-glycans from the protein in a gel by optimised peptide-N-glycosidase F digestion. The released glycans are then tagged with the fluorophore, 2-amino benzamide. The labelled glycan pools (containing 50-100 femtomoles of glycans) are resolved by predictive normal phase high performance liquid chromatography (HPLC) on an amide based column or by reverse phase HPLC on a C18 column. Preliminary structural assignments are confirmed by exoglycosidase array digestions of the entire glycan pool. Complementary matrix-assisted laser desorption/ionization-mass spectrometry, which requires 10-20 times as much sugar for a single run, can be used where there is sufficient material. This provides a composition analysis but not linkage information.

Structural elucidation of the N- and O-glycans of human apolipoprotein(a): role of o-glycans in conferring protease resistance.

Apolipoprotein(a) (apo(a)) is a multikringle domain glycoprotein that exists covalently linked to apolipoprotein B100 of low density lipoprotein, to form the lipoprotein(a) (Lp(a)) particle, or as proteolytic fragments. Elevated plasma concentrations of apo(a) and its fragments may promote atherosclerosis, but the underlying mechanisms are incompletely understood. The factors influencing apo(a) proteolysis are also uncertain. Here we have used exoglycosidase digestion and mass spectrometry to sequence the Asn (N)-linked and Ser/Thr (O)-linked oligosaccharides of human apo(a). We also assessed the potential role of apo(a) O-glycans in protecting thermolysin-sensitive regions of the polypeptide. Apo(a) contained two major N-glycans that accounted for 17% of the total oligosaccharide structures. The N-glycans were complex biantennary structures present in either a mono- or disialylated state. The O-glycans were mostly (80%) represented by the monosialylated core type 1 structure, NeuNAcalpha2-3Galbeta1-3GalNAc, with smaller amounts of disialylated and non-sialylated O-glycans also detected. Removal of apo(a) O-glycans by sialidase and O-glycosidase treatment dramatically increased the sensitivity of the polypeptide to thermolysin digestion. These studies provide the first direct sequencing data for apo(a) glycans and indicate a novel function for apo(a) O-glycans that is potentially related to the atherogenicity of Lp(a).

A sensitive mapping strategy for monitoring the reproducibility of glycan processing in an HIV vaccine, RGP-160, expressed in a mammalian cell line.

The external envelope glycoprotein (gp160) of HIV-1 is a candidate for vaccines against AIDS. Most of the surface of the molecule is shielded by carbohydrate and the structures and locations of these glycans may be important in defining the immunogenicity of the viral coat. Here we report a sensitive mapping strategy for profiling and analysing the N-glycosylation of gp160, based on chemical release of glycans, fluorescent labelling and HPLC analysis. This approach has been validated in terms of establishing the reproducibility of all steps in the analytical procedure and on overall reproducibility on a run-to-run and day-to-day basis. The validated analysis technique was used to monitor the consistency of N-glycosylation of one rgp 160 vaccine candidate produced in baby hamster kidney (BHK) cell culture. It was demonstrated that the variation in the glycan profiles of 6 different lots was not statistically significant.

Screening neutral and acidic IgG N-glycans by high density electrophoresis.

IgG carries bi-antennary N-linked glycans which differ in degrees of galactosylation, core fucosylation and bisecting N-acetyl glucosamine. The majority of these are non-sialyated closely related neutral structures which can be resolved by HPLC analysis, but which are difficult to separate in techniques such as fluorophore-coupled carbohydrate electrophoresis. Derivatisation with the singly charged fluorophore, 2-amino benzoic acid and separation in gels with a 30% monomer content in tris/glycine buffer enabled separation of neutral glycans. In particular, agalactosyl glycans with either a core fucose substitution or bisecting N-acetyl galactosamine could be resolved. Good separation of mono- and di-galactosylated glycans was also achieved with this system. It was shown that IgG can be separated from serum by size-exclusion and anion exchange chromatography with minimal contamination, with complete glycan release accomplished by the enzyme peptide-N-glycosidase F (F. meningosepticum). This method of resolving IgG glycans could be used to monitor patients in which glycosylation changes may have a diagnostic value, as in rheumatoid arthritis. It could also be used to monitor recombinant IgG glycosylation where routine screening is required in the biotechnology industry.

An indirect effect of an antibody on complement deposition and lysis of differently sensitized surrounding cells.

Lysis of papain-treated group A and B erythrocytes by human complement was studied by an anti-A (BRIC. 131) and an anti-B (BRIC. 30) IgM monoclonal antibody in 51Cr release assays. The indirect effect of membrane-bound antibody, i.e. its influence on complement binding to sensitized surrounding cells, was examined in a cold target competition test in which sensitized, non-labelled cells are present along with sensitized labelled cells and complement. The mode by which anti-A antibodies indirectly suppressed lysis of sensitized B cells up to 20-fold was studied by following C1q and C3b binding. C1q binding to both types of erythrocytes was not altered in mixed populations of erythrocytes in the presence of both antibodies. Binding of C3b to a mixture of both cell types was, however, suppressed, when both antibodies were present. C3b deposition in mixed cell populations did not reach a significantly higher extent than deposited to one type of erythrocyte alone. This was consistent with the results from competitive lysis and suggests that the anti-A captured most C3b at high anti-A concentrations and deprived the similarly sensitized B erythrocytes of complement. We think that this phenomenon is not due to an uneven removal of complement regulatory proteins from A and B erythrocytes by papain. Instead, the phenomenon might be due to an inherent property of anti-A mAb to better produce nucleation sites for C3 convertases which, upon binding factor B, better compete for the limiting factor D. A mathematical analysis of cold target competition experiment (containing 2430 individual measurements) also shows that the distribution of complement between the competing A and B erythrocyte population is uneven, since it predicts that in any given antibody combination the majority of complement is bound to A erythrocytes. This is consistent with the measured average percentage of lysis.

Red-cell bound anti-A is more efficient than anti-B in competition for fluid phase complement.

Lysis of group A and B erythrocytes by human complement was studied by an anti-A (BRIC.131) and an anti-B (BRIC.30) IgM monoclonal antibody in a 51Cr-release assay. The relative concentration of membrane-bound immunoglobulins was detected by flow cytometric analysis, and the amount of C1q and C3 bound to the sensitized red cells was measured by using purified, 125I-labelled molecules. The direct haemolysis was identical with both reagents in the presence of excess and suboptimal complement over a wide range of antibody concentration (between 50 and 7000 ng/ml). The indirect effect of membrane-bound antibody, i.e. its influence on complement binding by sensitized bystander cells, was examined in a cold target competition assay in which sensitized, non-labelled cells are present when complement is incubated with sensitized labelled cells. We have found that the competitive capacity of sensitized erythrocytes correlated with the amount of membrane-bound immunoglobulins. In accordance with our earlier findings, an equal level of target and competitor cell lysis was obtained only if the fluid phase anti-B antibody concentration was 2 to 4 times higher than that of the anti-A antibodies. We demonstrate in this paper that the different competitive activity of IgM anti-A and anti-B monoclonal antibodies might be accounted for by differences in their C1q and C3 binding capacities.

Interactions in the complement-mediated lysis of blood group AB erythrocytes sensitized simultaneously with anti-A and anti-B monoclonal antibodies.

The lysis of group AB erythrocytes by human complement was studied by different anti-A and anti-B IgM monoclonal antibodies (mabs) in a 51Cr-release assay. The concentration of membrane-bound immunoglobulin was detected by ELISA, and the amount of C1q and C3 bound to sensitized red cells was measured by using purified, 125I-labelled molecules. We have demonstrated that there is an exponential relationship between the concentration of the sensitizing IgM mabs and C1q binding to the sensitized AB cell. The efficiency of binding was related to the number of antibodies bound; thus, anti-A sensitized cells bound 3-6 times more C1q than anti-B sensitized cells did. AB cells, on the other hand, bound similar amounts of C3 whether anti-A or anti-B was present. The lytic efficiencies of the various IgM mabs during short incubation times were different, suggesting that the complement activation rates vary widely with different antibodies on the AB cell membrane. The binding of C1q to an antibody-sensitized target activates a cascade, whose components may migrate away from the sensitizing antibody; interactions between the activation processes generated by the anti-A and anti-B antibodies may thus occur. Choosing appropriate pairs of anti-A and anti-B mabs for the simultaneous sensitization of AB cells has indeed resulted in stimulation in some and inhibition in other combinations of mabs. It is suggested that stimulation is observed when the activated intermediates are produced in excess, whereas inhibition occurs when a shortage of activated intermediates prevents mutual utilization.

Biochemical studies on red blood cells from a patient with the Inab phenotype (decay-accelerating factor deficiency).

A 38-year-old Russian woman (KZ) has been identified as the fourth proposita with the Inab blood group phenotype. Like the first two propositi, she has a chronic intestinal disorder and, as shown for the third proposita, her Inab phenotype is demonstrably inherited. KZ's serum contained anti-IFC, which reacted with a red blood cell (RBC) membrane component with an Mr of 70,000, which is decay accelerating factor (DAF). Her RBCs lacked all Cromer-related blood group antigens and DAF. Her RBCs were no more susceptible than normal control RBCs to lysis in acid lysis or in rabbit or human antibody-initiated complement lysis tests. Northern blots of total RNA isolated from KZ's Epstein-Barr virus-transformed lymphoblasts showed a marked reduction of DAF mRNA when compared with normal. Polymerase chain reaction (PCR) amplification of cDNA confirmed this reduced level of DAF mRNA. Sequencing of the PCR product showed a 44-nucleotide deletion in the mRNA close to the short consensus repeats IIIa/IIIb intron/exon boundary. This deletion results in a change in the reading frame that places a termination codon six amino acids after the deletion. The putative translation product would lack a glycosyl phosphatidyl-inositol linkage site and, therefore, would not be membrane-bound in the RBC.

Protection by alpha-thalassaemia against Plasmodium falciparum malaria: modified surface antigen expression rather than impaired growth or cytoadherence.

We have attempted to determine the cellular mechanism by which alpha-thalassaemia may protect against Plasmodium falciparum malaria. Invasion and development of P. falciparum in the microcytic red cells of two-gene deletion forms of alpha-thalassaemia when measured morphologically or by [3H]hypoxanthine incorporation were normal compared to controls. Normal invasion rates were also observed following schizogony in thalassaemic red cells. Neither the addition of the oxidant menadione, 30% oxygen, nor modified medium, produced differential damage to parasites within thalassaemic cells. Furthermore, there were no significant differences in the binding of P. falciparum-parasitized alpha-thalassaemic and normal cells to C32 melanoma cells in vitro. However, when neoantigen expression on the surface of infected thalassaemic cells was estimated using a quantitative radiometric antiglobulin assay, clear differences were observed. It was found that alpha-thalassaemic cells bound higher levels of antibody from serum obtained from individuals living in a malaria endemic area than control normal red cells. The binding ratio for thalassaemic compared with controls was 1.69 on a cell-for-cell basis, and 1.97 when related to surface area. The binding of antibody from immune serum increased exponentially during parasite maturation. We also found increased binding of naturally occurring antibody present in non-immune serum to parasitized thalassaemic red cells which also increased during parasite maturation. We conclude that the protection afforded by thalassaemia against malaria may not reside in the ability of parasites to enter, grow or cytoadhere to endothelium in such cells, but may be related to immune recognition and subsequent clearance of parasitized red cells.

Surface antigen expression on Plasmodium falciparum-infected erythrocytes is modified in alpha- and beta-thalassemia.

In an attempt to determine the mechanism whereby thalassemia in its milder forms may protect against malaria, we have examined the expression of neoantigen at the surface of Plasmodium falciparum-parasitized thalassemic red cells. Neoantigen expression was estimated by measurement of antibody bound after incubation in serum from adults living in a malaria-endemic area, using a quantitative radiometric antiglobulin assay. We found that P. falciparum-parasitized alpha- and beta-thalassemic red cells bind greater levels of antibody from endemic serum than controls: mean binding ratios (+/- SE), respectively, for alpha- and beta-thalassemia compared with controls were 1.69 +/- 0.12 and 1.23 +/- 0.06 on a cell for cell basis, and 1.97 +/- 0.11 and 1.47 +/- 0.08 after a correction for surface area differences. Binding of antibody increased exponentially during parasite maturation. In addition, we found a small but significant degree of binding of naturally occurring antibody to parasitized red cells, the extent of which was also greater in thalassemia. The apparent protective effect of thalassemia against malaria may be related to enhanced immune recognition and hence clearance of parasitized erythrocytes.

Human monoclonal anti-D with reactivity against category DVI cells used in blood grouping and determination of the incidence of the category DVI phenotype in the DU population.

B-lymphoblastoid cell lines transformed by Epstein-Barr virus were produced from cells obtained from a hyperimmunised donor with serum anti-D activity against category DVI red cells and enriched for this activity by rosetting with category DVI red cells. Three clones produced IgG1 anti-D and had stable cell growth and continuous secretion of antibody in prolonged culture. The monoclonal antibodies reacted with category DVI red cells, when assessed manually and in an automated blood grouping system, and are useful blood grouping reagents for the detection of the category DVI phenotype. Using a radiometric technique, the number of antibody molecules bound to category DVI red cells from 5 individuals was estimated to range from 2,800 to 11,200 per cell. Five percent of blood donors classed as Du in the south western region were found to have the category DVI phenotype.

Heterogeneity in the ability of IgG1 monoclonal anti-D to promote lymphocyte-mediated red cell lysis.

Thirty-four IgG anti-D human monoclonal antibodies (mAb) derived from 18 donor were assessed for their ability to mediate lysis of D+ red cells by lymphocytes in antibody-dependent cell-mediated cytotoxicity assays. Cell-bound antibody was quantified and the mAb were compared at similar levels of sensitization. The majority (23/31) of IgG1 and all (3/3) IgG3 mAb were ineffective; two donors produced both lytic and non-lytic anti-D mAb. Greater sensitivity was achieved using fluid-phase antibody (as culture supernatants) in the assay than was obtained with pre-sensitized red cells. Minimum levels of 2000 anti-D molecules per cell were required for lysis using pre-sensitized cells. Partial D red cells (DIVa, DVa and DVI) were lysed by three mAb that were lytic with normal D+ cells. There was no relationship between lytic ability and Gm allotype or D epitope specificity of the antibodies. Four mAb to other blood group specificities were tested: two (anti-E and anti-G) were lytic and two (anti-c and anti-Kell) were not lytic. Possible reasons for the heterogeneity of the lytic activity by the mAb are discussed.

Studies on the sensitivity to complement-mediated lysis of erythrocytes (Inab phenotype) with a deficiency of DAF (decay accelerating factor).

No episodes of clinically significant in vivo haemolysis have been reported in individuals with a novel form of decay accelerating factor (DAF) deficiency (Inab phenotype), nor do functional in vitro assays for complement-mediated haemolysis show the extreme sensitivity to lysis characteristic of paroxysmal nocturnal haemoglobinuria (PNH) erythrocytes. DAF appears to be totally deficient in the Inab erythrocytes as judged by immunochemical and functional assays. Unlike PNH, the only other described DAF deficiency (where several other phosphatidylinositol (PI)-linked membrane proteins are also absent), the only protein lacking from Inab erythrocytes appears to be DAF. The Inab phenotype seems to be an inherited specific defect in DAF whereas PNH is an acquired defect in the mechanism of insertion of PI-linked proteins into cell membranes. These findings support the view that susceptibility of PNH erythrocytes to in vivo and in vitro complement-mediated haemolysis is not due simply to DAF deficiency but to either the combined lack of several membrane proteins or to deficiency of other regulatory proteins such as the membrane attack complex inhibitor/homologous restriction factor (MIP/HRF). The findings also raise questions as to the role of erythrocyte DAF.

Epitopes on sialoglycoprotein alpha: evidence for heterogeneity in the molecule.

The binding of 15 125I-labelled mouse monoclonal antibodies to cell-surface sialoglycoprotein alpha (SGP alpha: synonym Glycophorin A) was studied using intact IgG and Fab fragments. It was estimated that the number of sialoglycoprotein alpha (SGP alpha) molecules per red cell is of the order of 1 x 10(6) and the number of sialoglycoprotein delta (SGP delta; synonym Glycophorin B) molecules per red cell is of the order of 1.7-2.5 x 10(5). Competitive binding assays showed that antibodies of the same blood group specificity (four anti-Ns reacting with SGP alpha and SGP delta (BRIC 33, BRIC 115, BRIC 120, BRIC 123) and four anti-Wrbs reacting with SGP alpha (R7, BRIC 14, BRIC 89, BRIC 93) inhibited binding of each other to red cells. Two antibodies (R1.3 reacting with SGP alpha and SGP delta and R18 reacting with SGP alpha) recognized distinct epitopes, and the remaining five antibodies (BRIC 116, BRIC 117, BRIC 119, BRIC 127, R10 reacting with SGP alpha) partially inhibited binding of each other to red cells. This latter observation and the finding that four of these antibodies (BRIC 116, BRIC 117, BRIC 119, BRIC 127) bind to a considerably smaller number of antigen sites (1.69-2.71 x 10(5) for intact IgG) than the maximum value obtained, suggests heterogeneity of glycosylation within SGP alpha molecules. The functional affinities of the IgG antibodies ranged from 1 x 10(5) to 4 x 10(7)M-1.

Inhibition of malarial parasite invasion by monoclonal antibodies against glycophorin A correlates with reduction in red cell membrane deformability.

The effect of well-characterized monoclonal antibodies to red cell surface molecules on the invasion of human red cells by the malarial parasites Plasmodium falciparum and Plasmodium knowlesi was examined. Antibodies to glycophorin A (GP alpha) inhibit invasion for both parasite species, and this is highly correlated with the degree to which they decrease red cell membrane deformability as measured by ektacytometry. This effect on rigidity and invasion was also seen with monovalent Fab fragments. The closer the antibody binding site was to the membrane bilayer, the greater was its effect on inducing membrane rigidity and decreasing parasite invasion. Antibodies to the Wright determinant in particular were the most inhibitory. This differential effect of the various antibodies was not correlated with their binding affinities or the number of sites bound per cell. Antibodies to surface molecules other than GP alpha were without effect. A novel mechanism is described whereby monoclonal antibodies and their Fab fragments directed at determinants on the external surface of red cells might act to inhibit invasion by malarial parasites by altering membrane material properties.

Ability of monoclonal anti-D antibodies to promote the binding of red cells to lymphocytes, granulocytes and monocytes.

Red cells sensitized with IgG1 or IgG3 monoclonal anti-D antibodies were used in rosette assays with human lymphocytes, monocytes and granulocytes. With all three cell types. IgG3 antibodies promoted a greater degree of rosette formation than IgG1 antibodies. Monocytes required a minimum of about 0.5 x 10(3) IgG3 molecules per red cell for rosette formation, and granulocytes and lymphocytes required around 1 x 10(4) IgG3 molecules per red cell. Approximately 80% of monocytes and granulocytes and 10% of lymphocytes were capable of rosette formation. These results are consistent with differences in the number and affinity of Fc receptors on different leucocytes. When compared with previous data these results suggest that binding of monocytes to monoclonal anti-D sensitized red cells is very similar to that of red cells sensitized with polyclonal antisera. Lymphocytes and granulocytes, however, appear to bind less well to red cells sensitized with certain monoclonal antibodies than with polyclonal antibodies. These findings may be of relevance to the prophylactic use of monoclonal anti-D antibodies.

An in-vitro assessment of the functional activity of monoclonal anti-D.

The response of human monocytes to red cells sensitized with known levels of monoclonal antibody to the Rh antigen D (anti-D) was compared with that of polyclonal anti-D. Monocyte response was determined by measuring red cell adherence, erythrophagocytosis, monocyte-mediated red cell lysis and luminol-dependent chemiluminescence. By all criteria, monoclonal and polyclonal antibodies showed comparable activity, with IgG3 antibodies promoting a greater monocyte-red cell interaction than IgG1 antibodies. It is suggested that monoclonal anti-D may be effective in the prophylaxis of haemolytic disease of the newborn, providing such material is clinically acceptable.

Rate of interaction of IgG1 and IgG3 sensitized red cells with monocytes in the phagocytosis assay.

Another aspect of the preferential binding to monocytes of IgG3-coated erythrocytes over IgG1-coated erythrocytes has been demonstrated by measuring the rate of interaction in the phagocytosis assay. Erythrocytes sensitized with comparable numbers of IgG1 and IgG3 monoclonal and polyclonal anti-D antibodies were incubated with a monocyte preparation for up to 2 h. Interaction with the erythrocytes sensitized with monoclonal or polyclonal IgG3 was much more rapid than with erythrocytes sensitized with IgG1. With IgG3-sensitized erythrocytes maximum interaction was found after 30 min, whereas with IgG1-sensitized erythrocytes 2 h were required for maximum interaction. These results are consistent with the longer hinge region of IgG3 allowing bridges to be formed more easily between the negatively charged erythrocytes and monocytes.