Inhibition of processing of asparagine-linked carbohydrate chains on IgG2a by using swainsonine has no influence upon antibody effector functions in vitro

Removal of asparagine (Asn)-linked carbohydrate chains from IgG antibody molecules reduces their antibody effector functions such as C activation and FcR binding. We have prepared IgG2a mAb with modified structure of carbohydrate chains by treating the hybridoma cells with swainsonine, which inhibits the processing of Asn-linked carbohydrate chains at the site of action of mannosidase II. These antibodies have obtained the capacity to bind lentil lectin and have become sensitive to endoglycosidase H digestion, indicating the structural changes of oligosaccharides from complex type to hybrid type. They behaved in an identical manner to the normal IgG2a antibodies with regards to extracellular secretion, Ag-binding capacity, C-mediated hemolysis and FcR-mediated functions. Critical moieties of Asn-linked carbohydrate chains on IgG molecules to retain their antibody effector functions were discussed.     

Three M components IgA lambda, + IgG kappa n + IgG kappa h in one patient. II. Structurally different kappa n and kappa h chains associated with H(gamma) chains sharing idiotypic determinants

Structural analysis of purified IgG kappa h and IgG kappa n molecules of DA myeloma paraproteins indicated that the two IgG had different electrophoretic mobility and that L-kappa h had an unusually heavy m.w. (30,000). Peptide mapping showed the existence of additional peptides in the L-kappa h map when compared to the L-kappa n map. Total amino acid analysis showed that L-kappa h contained two additional cysteine residues and certain other amino acids than L-kappa n contained. Sequence of the first 25 NH2-terminal amino acids showed differences at positions 4, 5, 15, 18, and 21, but both L-kappa h and L-kappa n belong to the same V kappa IV subgroup. IgG kappa n but not IgG kappa h reacted with anti-gamma 1 antiserum, indicating that H chains of these two paraproteins were also different. Sera from rabbits immunized with IgG kappa n or IgG kappa h, and rendered specific for idiotypic determinants by appropriate absorption, were used for idiotype characterization of these components. Immunodiffusion, immunoelectrophoresis, and direct hemagglutination demonstrated that IgG kappa h and IgG kappa n cross-reacted partially. Inhibition tests disclosed that the main anti-idiotypic antibody was directed against a conformation structure of the complete IgG kappa h molecule, whereas cross-reaction was due to partial idiotypic similarity between H chains of the IgG kappa h and IgG kappa n. In addition, each of these paraproteins seemed also to bear private idiotypes on their H and L chains.     

Murine plasma cells secreting more than one class of immunoglobulin heavy chain. II. SAMM 368--a plasmacytoma secreting IgG2b-kappa and IgA-kappa immunoglobulins which do not share idiotypic determinants.

SAMM 368 is a BALB/c plasmacytoma which secretes IgG2b-kappa and IgA-kappa paraproteins. Immunofluorescence studies of ascites cells from the tumor with purified, heavy chain class-specific antiglobulins demonstrate that single cells contain both IgG2 and IgA heavy chains. Idiotypic antisera prepared in mice and rabbits indicate that the two paraproteins produced by the tumor do not share idiotypic determinants. Analysis of the purified paraproteins for allotype markers showed that SAMM 368 IgA bears the BALB/c A12,13,14 determinants. SAMM 368 IgG2b does not carry any detectable allotypic determinants in spite of the fact that heterologous antisera identify the paraprotein as IgG2b.     

Shared N-terminal sequences in monclonal IgMkappa and IgGkappa proteins from a patient with a complex multiple paraprotein disorder.

The N-terminal sequence analyses were performed on the heavy (H) and light (L) chains of the idiotypically identical IgM kappa and IgG kappa paraproteins isolated from the serum of patient, Cam. The N-terminal 39 residues of the kappa chains of the IgM and IgG were identical and belonged to the human V kappa III subgroup. This sequenced stretch included the first L chain hypervariable region. The N-terminal 27 residues of the variable regions (VH) of the respective mu and gamma heavy chains were also identical and belonged to the human VHIII subgroup. These identical VH sequences were unique with lysine residues at positions 13 and 19. This dual lysine substitution has not been seen in 37 other human VHIII sequences reported in the literature. This N-terminal sequence homology in the V-regions of Cam IgM kappa and IgG kappa paraproteins and the shared idiotypy expressed by Cam IgM, IgG, and IgA proteins strongly suggest the existence of complete structural homology in the variable regions of the and L chains of these Ig molecules of three separate Ig classes. At the cellular and genetic level, these results point toward a common clonal origin for the idiotypically related Ig molecules and suggest that identical V-region (VH and VL) genes were utilized by the Cam lymphoid clone in the biosynthesis of the respective IgM, IgC, and IgA proteins.     

Study of the subclasses of immunoglobulin G. II. Qualitative and quantitative characteristics of IgG subclasses using antisera systems]

Bispecific antisera, or "antisera-systems", containing class- and subclass-specific antibodies to IgG were obtained from rabbits, goats and guinea pigs after brief courses of immunization with purified G1, G2, G3 and G4 paraproteins. After the elimination of antibodies to light chains by adsorption these antisera were tested in immunoelectrophoresis and radial immunodiffusion in gel with sera containing G paraproteins of different subclasses. In immunoelectrophoresis double lines and in radial immunodiffusion with G paraproteins of heterologous subclasses double rings were obtained: the external lines (or the external rings) were formed as a result of interaction between G paraproteins and antibodies to class-specific IgG determinants, the inner lines (or the inner rings) were formed as a result of interaction between the corresponding subclass of normal IgG and subclass-specific antibodies. The identification of different G paraprotein subclasses gave similar results when carried out with "antisera-systems" and with monospecific antisera to the corresponding IgG subclasses. "Antisera-systems" proved to be suitable for use in the identification of G paraprotein subclasses, as well as in the quantitation of different subclasses in normal IgG.     

Density comparisons of heavy chains of membrane and secreted immunoglobulins of mouse.

In order to explore structural differences between membrane and secreted immunoglobulins the buoyant densities of mouse immunoglobulin (Ig) heavy (H) chains were compared by isopycnic centrifugation in CsCl containing guanidine hydrochloride. The buoyant densities, under denaturing conditions, of mouse myeloma protein MOPC 21 IgG, MOPC 315 IgA and MOPC 104E IgM H chains were consistent with their carbohydrate contents. Mouse membrane IgM and MOPC 104E-secreted IgM H chains were of equal density. The buoyant densities of MOPC 104E-secreted IgM and spleen-cell-secreted IgM H chains were indistinguishable. The IgD-like membrane H chain was denser than membrane IgM H chain, and its carbohydrate content was calculated to be 15.5%. The resolution of the technique was sufficient to conclude that the apparent 1500 mol.wt. difference, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, between membrane and secreted IgM H chains was due to peptide rather than to carbohydrate. The results also imply that intact membrane IgM and IgD bind detergent and are thus integral membrane proteins.     

Structural analysis of carbohydrate chains of normal and myeloma immunoglobulins G using HPLC

A comparative analysis of carbohydrate chains from human normal IgG and myeloma IgG (subclass 4) was carried out using HPLC. It is shown that both IgGs contain the same carbohydrate chains (biantennary and bisected) but the relative amount of bisected and incomplete chains (with fewer terminal galactose residues) is higher in myeloma IgG4. Two earlier unknown minor oligosaccharides were isolated from normal IgG and structurally studied by means of the partial hydrolysis and the Smith degradation.     

A comparative study of the N-linked oligosaccharide structures of human IgG subclass proteins.

Quantitative oligosaccharide profiles were determined for each of 18 human IgG paraproteins representing the four subclasses. Each paraprotein exhibits a unique profile that may be substantially different from that observed for polyclonal IgG. The IgG2 and some IgG3 proteins analysed exhibit a predominance of oligosaccharide moieties having galactose on the Man(alpha 1----3) arm rather than the Man(alpha 1----6) arm; it was previously held that galactosylation of the Man(alpha 1----6) arm is preferred, as observed for IgG1, IgG4 and polyclonal IgG. An IgG4 protein is reported that has galactosylated Man(alpha 1----3) and Man(alpha 1----6) arms on both Fc-localized carbohydrate moieties; previous findings suggested that such fully glycosylated structures could not be accommodated within the internal space of the C gamma 2 domains. Unusual monoantennary oligosaccharides present in IgG2 and IgG3 proteins were isolated and their structures determined.     

Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region

Chimeric mouse-human IgG was used to study the structural and functional roles of the carbohydrate present in the CH2 domain of human IgG molecules. To remove this N-linked carbohydrate, Asn-297, the oligosaccharide attachment residue, was changed to either Gln (a conservative replacement) or His for IgG1 or Lys for IgG3 (nonconservative replacements) by site-directed mutagenesis. Carbohydrate-deficient antibodies are properly assembled and secreted and bind Ag and protein A. However, aglycosylated IgG are more sensitive to most proteases than their corresponding wild-type IgG, indicating some conformational changes have occurred. Aglycosylated IgG do not bind to the human Fc gamma RI and do not activate C; depending on the isotype, C1q binding ability is either completely lost (IgG1) or dramatically decreased (IgG3). The serum half-life in mice of aglycosylated IgG1-Gln remains the same as wild-type IgG1, 6.5 +/- 0.5 days, whereas aglycosylated IgG3-Gln has a shorter half-life, 3.5 +/- 0.2 days, compared to that of wild-type IgG3, 5.1 +/- 0.4 days. These results indicate the carbohydrate interposed between CH2 domain of human IgG is necessary to maintain the appropriate structure for the maintenance of many of the effector functions dependent on the CH2 domain.     

Monomeric complement-activating IgG paraproteins

Three patients presented a unique syndrome of recurrent panniculitis with an IgGkappa paraprotein and depletion of the early components of the classical pathway of complement. The IgGkappa paraproteins were monomers with a normal structure, and with no evidence for aggregation, as assessed by electron microscopy and ultracentrifugation. Both heavy and light chains were of normal molecular size (SDS-PAGE), and the paraproteins were not heavily glycosylated. However, the paraproteins from all three patients had unusual features that included abnormal behavior on gel filtration chromatography and a heavy chain of high pI. When analyzed by fast protein liquid chromatography (Superdex 200), elution of the paraproteins was retarded, particularly when the ionic strength was increased. This retardation was partially reversed in 20% alcohol, and fully reversed in 6 M guanidine-HCl. Neither anti-C1 inhibitor nor anti-C1q autoantibodies were found in any of the patients' sera. However, the paraproteins bound to the globular heads of C1q at normal ionic strength. They activated C4 in normal human serum, but not in C1q-deficient serum. Activation led to the formation of C1s-C1 inhibitor complexes. Taken together, the data suggest that the unusual paraproteins have the capacity to bind C1q, which then leads to activation of C1. The ability of these paraproteins to activate C1, in spite of their being soluble monomers, is likely to be related to their unique physicochemical features.     

Incidence of three cross-reactive idiotypes on human rheumatoid factor paraproteins

The basis for rheumatoid factor (RF) production in autoimmune or lymphoproliferative diseases cannot be understood without defining the molecular factors that dictate RF structure and specificity. Recently three different mAb (6B6.6, 17.109, and G6) have been developed that define cross-reactive idiotypes (CRI) on intact L or H chains of human monoclonal RF cryoglobulins. However, the true incidence of these CRI among RF and their relationship to each other have not been delineated. In the present experiments, a panel of 163 randomly selected IgM paraproteins was evaluated for the expression of the two kappa L chain CRI, 6B6.6 and 17.109, and the H chain CRI, G6. Among the paraproteins with kappa L chains, 14% expressed the 17.109 CRI, and 9% expressed the 6B6.6 CRI. Both ELISA and Western immunoblotting experiments showed that the two L chain CRI were mutually exclusive. Anti-IgG activity was documented in 22 of the IgM-kappa paraproteins, among which mAb 6B6.6 reacted with 7 (32%) and mAb 17.109 with 6 (27%). Both CRI were expressed exclusively by L chains within the kappaIII variable gene subgroup. Although 17.109 CRI+ paraproteins had kappaIIIb L chains, none of the 6B6.6 CRI+ paraproteins possessed L chains with this kappa sub-subgroup specific Ag. The G6 CRI was found predominantly among RF paraproteins and was frequently yet not exclusively associated with the 17.109 CRI+ L chains. Additional experiments were performed on a panel of normal adult human sera and documented the presence of 6B6.6 and 17.109 CRI on a small percentage (0.1 to 2.0%) of IgM from most individuals. These data indicate that 1) the mAb 6B6.6 and 17.109 identify two major and distinct CRI among IgM-RF paraproteins, 2) both CRI are associated exclusively with kappaIII L chains, 3) kappaIIIb and kappaIII non-b L chains are equally prevalent among IgM-RF, 4) the G6 H chain CRI is frequently associated with 17.109 CRI+ L chains, but not with 6B6.6 CRI+ L chains, and 5) although the ability to make 6B6.6 and 17.109 CRI+ kappa L chains is common in humans, these CRI are present in low concentrations in normal IgM.     

Glycosylation is necessary for the correct folding of human immunodeficiency virus gp120 in CD4 binding.

Conflicting results have been reported regarding the role of carbohydrate on human immunodeficiency virus (HIV) envelope glycoprotein gp120 in CD4 receptor binding. Glycosylated, deglycosylated, and nonglycosylated forms of HIV type 1 (HIV-1) and HIV-2 gp120s were used to examine CD4 receptor-binding activity. Nonglycosylated forms of gp120 generated either by deletion of the signal sequence of HIV-1 gp120 or by synthesis in the presence of tunicamycin failed to bind to CD4. In contrast, highly mannosylated gp120 bound to soluble CD4 molecules well. Enzymatic removal of carbohydrate chains from glycosylated gp120 by endoglycosidase H or an endoglycosidase F/N glycanase mixture had no effect on the ability of gp120 to bind CD4. An experiment which measured the ability of gp120 to bind to CD4 as an assay of the proper conformation of gp120 showed that carbohydrate chains on gp120 are not required for the interaction between gp120 and CD4 but that N-linked glycosylation is essential for generation of the proper conformation of gp120 to provide a CD4-binding site.     

Lymphocytic plasmocytoid lymphoma with a three-banded gammopathy: reactivity of one of these paraproteins with cytomegalovirus

We report the case of a 70-year-old woman suffering from small lymphocytic, plasmocytoid lymphoma with abdominal lymphomas and infiltration of the lung and the bone marrow. A three-banded, IgG lambda, IgM lambda and IgA lambda-paraproteinemia was determined using immunofixation. Because of the patient's high antibody titre against cytomegalovirus (CMV), the possible reactivity of these paraproteins with CMV was studied. The immunoglobulins were transferred to nitrocellulose sheets by a contact diffusion blotting system. CMV was applied to these sheets and the IgG lambda-paraprotein was shown to bind CMV. The reactivity of only one of the paraproteins with CMV suggests an oligoclonal origin of this gammopathy. In addition to the malignant disease an abnormal immune response to a CMV infection could be the cause of this three-banded gammopathy.     

Cobra venom factor and human C3 share carbohydrate antigenic determinants

As tools to study structural relationships of cobra venom factor (CVF) and human complement component C3, murine monoclonal antibodies to CVF were produced. In this paper we describe two of these monoclonal anti-CVF antibodies designated GV1.8 and GV1.10, both of which bind to carbohydrate epitopes. On immunoblotting, antibody GV1.8 binds to both the alpha- and beta-chains of CVF, whereas antibody GV1.10 binds only to the alpha-chain of CVF. After enzymatic deglycosylation of CVF with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase), both antibodies lose their ability to bind to the deglycosylated protein. Additionally, the free oligosaccharide chains of CVF are able to inhibit the binding of antibodies GV1.8 and GV1.10 to CVF on enzyme-linked immunosorbent assay, further demonstrating their carbohydrate specificity. Both monoclonal antibodies to CVF cross-react with human C3. Antibody GV1.8 binds to both chains of human C3 indicating that the shared antigenic epitope present on the two glycosylated chains of CVF is also present on the two chains of human C3. Antibody GV1.10 cross-reacts only with the beta-chain of human C3 which is the homologous chain to the alpha-chain of CVF. After enzymatic deglycosylation of human C3 by N-glycanase, both antibodies lose their ability to bind to the deglycosylated protein consistent with the carbohydrate nature of the recognized epitopes. These results indicate that CVF and human C3 share carbohydrate epitopes on their homologous and nonhomologous chains.     

IgA protease activity of microbes in the genus Bordetella

Monoclonal IgA paraproteins of subclasses 1 and 2, isolated from the sera of myeloma patients, were incubated for 4, 24, 48 and 72 hours with B. pertussis, B. parapertussis, B. bronchiseptica cultures, as well as Haemophilus influenzae strain. The fragmentation of IgA was studied by immunielectrophoresis with antisera to alpha-chain, to Fab alpha + Fc alpha, to Fab alpha and with antisera to light chains corresponding to the type of paraprotein. B. pertussis and B. parapertussis were found to have subclass-unspecific IgA protease which splitted off a cathode fragment, similar to Fab-fragment and, probably, corresponding to the variable domain of alpha-chain (Fv), after 48-hour incubation. Similar IgA protease was detected in H. influenzae, found to have classical IgA1 protease as well. All Bordetella species under study splitted off anode components from IgA paraproteins of both subclasses. These components, containing the determinants of heavy and light IgA chains, were either IgA - alpha I-antitrypsin complexes or some IgA fragments with high electrophoretic motility. None of the strains under study splitted monoclonal IgG.     

Characterization of anti-heparan sulfate phage display antibodies AO4B08 and HS4E4

Heparan sulfates (HS) are linear carbohydrate chains, covalently attached to proteins, that occur on essentially all cell surfaces and in extracellular matrices. HS chains show extensive structural heterogeneity and are functionally important during embryogenesis and in homeostasis due to their interactions with various proteins. Phage display antibodies have been developed to probe HS structures, assess the availability of protein-binding sites, and monitor structural changes during development and disease. Here we have characterized two such antibodies, AO4B08 and HS4E4, previously noted for partly differential tissue staining. AO4B08 recognized both HS and heparin, and was found to interact with an ubiquitouys, N-, 2-O-, and 6-O-sulfated saccharide motif, including an internal 2-O-sulfate group. HS4E4 turned out to preferentially recognize low-sulfated HS motifs containing iduronic acid, and N-sulfated as well as N-acetylated glucosamine residues. Contrary to AO4B08, HS4E4 did not bind highly O-sulfated structures such as found in heparin.     

Evidence for shared idiotypy expressed by the IgM, IgG, and IgA serum proteins of a patient with a complex multiple paraprotein disorder.

The results of a comparative idiotypic analysis of multiple Ig paraproteins isolated from the serum of an individual patient, Ca, with Sj?gren's syndrome and Waldenstr?m's macroglobulinemia are reported. At initial presentation, Ca serum was found to contain two major paraproteins, an IgMkappa and an IgGkappa, together with a small elevation in the level of IgA protein. The patient's clinical course was characterized by dramatic and opposing changes in the respective serum levels of the IgMkappa and IgGkappa paraproteins over an extended time period that coincided in part with received chemotherapy. Idiotypic antigenic analysis of the IgMkappa and IgGkappa paraproteins revealed that the two monotypic proteins shared identical idiotypic determinants. The Ca IgA serum fraction, specifically isolated by an immunoabsorbent and free of any IgG and IgM, was shown to possess idiotypic determinants identical to the IgG and IgM proteins. In extensive tests of specificity, the idiotypic determinants shared by Ca IgM, IgG, and IgA proteins were not present in large excesses of heterologous IgM and IgG, nor on Ig molecules contained in a large number of normal and myeloma sera.     

Structural analysis of N-linked oligosaccharides by a combination of glycopeptidase, exoglycosidases, and high-performance liquid chromatography

A simple, sensitive, and rapid method for the analysis of structures of N-linked carbohydrates is reported. The method involves four steps: preparation of carbohydrate chains from glycopeptides by N-oligosaccharide glycopeptidase digestion; derivatization of the reducing ends of carbohydrate chains with a fluorescent reagent, 2-aminopyridine, by using sodium cyanoborohydride; separation of oligosaccharide derivatives by reverse-phase high-performance liquid chromatography; and structural analysis of oligosaccharides by sequential exoglycosidase digestion. The elution positions of 50 standard oligosaccharide derivatives were determined by HPLC. The structure of an unknown oligosaccharide can be characterized by comparison of its elution position with those of the standard compounds. The method was applied to elucidate the structures of oligosaccharides in the myeloma IgG protein, Yot.     

Myelin-Associated Glycoprotein Shares an Antigenic Determinant with a Glycoprotein of Human Melanoma Cells

A sulfated 100K-dalton glycoprotein has been shown to be released into the culture medium of melanoma cells. Monoclonal antibodies 10C5 and 11B5, which were raised to human melanoma cells, as well as HNK-1 bind to this glycoprotein. It is shown here that mouse anti-myelin-associated glycoprotein (MAG) carbohydrate antibodies raised to human MAG and a human IgM paraprotein associated with neuropathy also bind to the same 100K molecule. However, anti-MAG antibodies recognizing peptide epitopes do not appear to react with this glycoprotein of melanoma cells, a result suggesting that its similarity to MAG is restricted to shared carbohydrate moieties. The anti-melanoma antibodies (10C5 and 11B5) resemble HNK-1 in binding to MAG and to some 19-28K-dalton glycoproteins and sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, the anti-melanoma antibodies cross-react with neural cell adhesion molecule (N-CAM), an observation emphasizing the shared antigenicity between MAG and other adhesion molecules. The results demonstrate that the anti-melanoma antibodies fall into a class of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, anti-human MAG antibodies, and L2 antibodies) that are characterized by reactivity against related carbohydrate determinants shared by human MAG, N-CAM, and several protein and lipid glycoconjugates of the PNS.