Two monoclonal antibodies highly specific for the blood group N determinant

Two monoclonal IgM antibodies, 179K and 35/5F, obtained following immunization of mice with A₂,MN or O,MN human erythrocytes, agglutinate NN and MN red cells strongly, and MM erythrocytes weakly. As shown by hemagglutination inhibition and solid phase ELISA, both antibodies are highly specific for the blood group N determinant. They react with N glycoprotein, its amino-terminal glycopeptides and with Ss glycoprotein (glycophorin B), which carries the blood group N determinant. They fail to react with M glycoprotein, M glycoprotein-derived glycopeptides, or with internal glycopeptides derived from N glycoprotein. Reaction of the antibodies with N glycoprotein is abolished by desialylation, periodate oxidation/borohydride reduction, orN-acetylation of the glycoprotein. Thus, the antibodies are specific for an epitope which includes sialylated oligosaccharide chain(s) and is located in the region of the amino-terminal leucine residue of N glycoprotein. MMU^– erythrocytes, lacking both blood group N and Ss glycophorin are non-reactive. Agglutination of MMU⁺ erythrocytes by the anti-N antibodies occursvia interaction with glycophorin B and correlates with the Ss phenotype of red cells MM,S erythrocytes are usually more strongly, agglutinated than MM,ss cells. The agglutination of MM erythrocytes decreases markedly as the pH is increased from 6 to 8, while agglutination of NN red cells is much less affected by shifts in pH over this range. As a result, both monoclonal antibodies are highly anti-N specific typing reagents when the agglutination assay is carried out at pH 8.     

Miltenberger Class I and II erythrocytes carry a variant of glycophorin A.

The membranes from Miltenberger Class I (Mi I) and II (Mi II) erythrocytes, two rare variants at the blood group MNSs locus, exhibited an abnormal glycoprotein of 32 kDa apparent molecular mass sharply stained by the periodic acid/Schiff procedure and a decreased content of glycoprotein alpha (synonym glycophorin A, glycoprotein MN) as seen on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Purified 125I-labelled Vicia graminea lectin binds to the unusual 32 kDa glycoprotein separated from Mi I and Mi II erythrocyte membrane of blood group NN or MN, but no significant labelling of this band was observed with Mi samples typed MM. On the basis of such lectin-labelling experiments we have described two heterozygous MN, Mi I individuals that carry one copy of an M gene producing a normal alpha-glycoprotein with M-specificity and one copy of a MiI gene producing a 32 kDa glycoprotein with N-specificity. Further investigations have shown that the 32 kDa glycoprotein was immunoprecipitated by two mouse monoclonal antibodies (R18 and R10) reacting specifically with the external domain of glycoprotein alpha. These results demonstrate that Mi I and Mi II erythrocytes carry an unusual variant of glycoprotein alpha.     

High frequency antigens of human erythrocyte membrane sialoglycoproteins, III. Studies on the EnaFR, Wrb and Wra antigens

The nature of the common erythrocyte antigens EnaFR and Wrb, that are both absent from En(a-) cells, and the rare Wra receptor, apparently encoded by an allele of Wrb, was investigated. Various modification, fractionation or cleavage products of erythrocyte membranes were used in hemagglutination inhibition assays. The EnaFR and Wrb antigens were shown to represent labile structures within the residues approx. 62-72 of the major (MN) sialoglycoprotein that require lipids, at least for complete expression of antigenic activity. During the course of these experiments, the arrangement of the MN glycoprotein's peptide chain with respect to the lipid bi-layer was also studied, using various proteinases. Furthermore, the MN glycoprotein was found to aggregate with the major membrane protein (band 3) in the presence of Triton X-100. The Wra antigen was shown to exhibit properties that differ considerably from those of the Wrb receptor. Analyses on the MN glycoprotein, isolated from the red cells of the only known Wra homozygote and two WraWrb individuals, did not reveal any amino-acid exchange within the residues 40-96 of the molecule. Therefore, the Wr locus that determines the presence or absence of the Wrb antigen on the MN glycoprotein might influence the post-translational modification of amino-acid residues, the structure of tightly bound lipids or the aggregation of the MN glycoprotein with a different protein such as band 3.     

Pj variant, a new hybrid MNSs glycoprotein of the human red-cell membrane.

An unusual glycoprotein variant (Pj) was found inherited through a caucasian family exhibiting atypical N and Nvg blood-group reactivities. Pj erythrocytes are blood-group-MS homozygous and have a normal sialic acid content. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the variant contains a new component Pj of 24kDa apparent molecular mass in the monomeric state which is sharply stained by periodic acid/Schiff reagent. Both blood-group-MN (alpha) and -Ss (delta) glycoproteins were present. Homodimers (Pj2) as well as heterodimers with MN-glycoprotein (alpha Pj) and the Ss-glycoprotein (delta Pj) were also identified. The new sialoglycoprotein Pj is trypsin- and chymotrypsin-resistant in situ and carries N- and Nvg- but not M- and S-reactivities. The Pj component is labelled by lactoperoxidase-catalysed radioiodination. A 3H label is also easily introduced into the sialic acid or the galactose and galactosamine of the Pj glycoprotein. It is proposed that the Pj is a hybrid glycoprotein containing the N-terminal end of delta-glycoprotein and the C-terminal end of the alpha-glycoprotein. This proposal is supported by the finding that Pj carries a leucine residue at its N-terminus and is not immunoprecipitated by a monoclonal mouse antibody (R18) reacting specifically with the external domain of glycoprotein alpha. The red cells from the proposita Pj were found positive for a very low frequency MN antigen named Sta.     

Human blood-group MN and precursor specificities: structural and biological aspects

The human blood-group MM and NN antigens carry 2 to 4 immunodominant groupings per repeating subunit and differ only by one sialic acid residue per immunodominant group. This residue covers in the MM antigen the β-D-galactopyranosyl group that is terminal in the N immunodominant structure and that, together with a terminal α-linked N-acetylneuraminic acid residue, is responsible for N specificity. M specificity was readily converted into N specificity by mild acid treatment. N structure is the immediate biochemical precursor of M structure, and M and N antigenic specificities are not determined by two allelic genes as believed hitherto. The NN antigen was inactivated by β-D-galactosidase as well as by removal of N-acetylneuraminic acid. Some of the reactivities of the NN antigen, lost upon β-D-galactosidase treatment, reappeared on subsequent partial N-acetylneuraminic acid removal. The structure uncovered by complete sialic acid depletion of MN antigens is the Thomsen—Friedenreich T antigen, the specificity of which is determined by β-D-galactopyranosyl groups. β-D-Galactosidase treatment transformed the T antigen into one possessing Tn activity. The significance of blood-group MN active substances extends to human breast cancer, where MN antigens were found in benign and malignant glands, but some of their precursors in cancerous tissue only.     

Structure of the Ss blood group antigens, II: a methionine/threonine polymorphism within the N-terminal sequence of the Ss glycoprotein

The N-terminal amino acid sequence (residues 1--35) of the Ss sialoglycoprotein (or glycophorin B) from human erythrocyte membranes of defined Ss blood group activity was determined by manual sequencing methods, using N-terminal tryptic or chymotryptic glycopeptides and various secondary peptides. The proposed structure differs considerably from that suggested on the basis of work with glucopeptides of unknown Ss blood group activity (Furthmayr, Nature 271, 519--523, 1978). Only one difference between glycopeptides from Ss and ss erythrocytes was found, i.e. a methionine/threonine polymorphism at position 29. On the basis of previous work (Dahr et al., Hoppe-Seyler's Z. Physiol. Chem. 361, 145--152, 1980), it is concluded that this amino acid heterogeneity represents the Ss polymorphism rather than the UX or UZ polymorphisms, which are in strong genetic linkage disequilibrium with the Ss antigens. A part of the sequence (residues 9--30) of the major (MN) red cell membrane sialoglycoprotein (glycophorin A) was re-investigated and revised at positions 11 and 17. As judged from the present data, the first 26 residues of the Ss and the blood group N-specific MN glycoprotein are identical. The sequence 27--35 of the Ss glycoprotein shows a homology with the residues 56--64 and 59--67 of the MN glycoprotein. Data on the partial N-terminal sequence of glycopeptides from a third erythrocyte membrane sialoglycoprotein (component D or glycophorin C) indicate that its structure is different from those of the two other glycoproteins.     

Common precursors of human blood group MN specificities

Human blood group MM and NN specific structures have the same precursors. Complete sialic acid removal produced the Thomsen-Friedenreich T antigen which was transformed into Tn antigen by $\text{E. coli}\text{β-}\text{D}$-galactosidase on red cells as well as on isolated T antigen. MN antigens and their precursors are most clearly defined by isologous human antisera.     

Different N-terminal amino acids in the MN-glycoprotein from MM and NN erythrocytes

Summary The major human erythrocyte membrane (MN-) sialoglycoprotein was purified from MM, MN, and NN cells using detergent gel and ion exchange chromatography. N-terminal analyses with dansyl-chloride revealed serine in preparations from MM and leucine in those from NN erythrocytes, whereas glycoprotein isolated from MN cells contained both the above amino acids. These data strongly suggest that the above residues may represent the structural difference between the M and N antigens. Evidence was also obtained that the Ss-glycoprotein, which is associated with N activity, exhibits the same N-terminal amino acid (leucine) as the MN glycoprotein from MN cells.     

Isolation and characterization of the human adenocarcinoma-associated glycoprotein gp40

The human adenocarcinoma-associated antigen gp40 is a cell surface glycoprotein recognized by murine monoclonal antibody KS1/4. A KS1/4-Sepharose affinity matrix was utilized to purify gp40 from detergent lysates of either tissue culture cells or nude mouse xenograft tumors of the human lung adenocarcinoma cell line P3-UCLA. This single immunoaffinity chromatography step yielded an antigen preparation of approximately 95% purity which was further characterized by immunochemical and enzymatic techniques. The gp40 molecule was shown to have both complex and high-mannose oligosaccharides comprising some 16% of the apparent molecular weight. The antigen preparation was suitable for gas-phase N-terminal amino acid sequencing and the first 16 residues of the N-terminus were determined. Despite considerable molecular heterogeneity, gp40 shows a single N-terminal sequence.     

Preserved MHC Class II Antigen Processing in Monocytes from HIV-Infected Individuals

Background

MHC-II restricted CD4+ T cells are dependent on antigen presenting cells (APC) for their activation. APC dysfunction in HIV-infected individuals could accelerate or exacerbate CD4+ T cell dysfunction and may contribute to increased levels of immunodeficiency seen in some patients regardless of their CD4+ T cell numbers. Here we test the hypothesis that APC from HIV-infected individuals have diminished antigen processing and presentation capacity.

Methodology/Principal Findings

Monocytes (MN) were purified by immuno-magnetic bead isolation techniques from HLA-DR1.01+ or DR15.01+ HIV-infected and uninfected individuals. MN were analyzed for surface MHC-II expression and for antigen processing and presentation capacity after overnight incubation with soluble antigen or peptide and HLA-DR matched T cell hybridomas. Surface expression of HLA-DR was 20% reduced (p<0.03) on MN from HIV-infected individuals. In spite of this, there was no significant difference in antigen processing and presentation by MN from 14 HIV-infected donors (8 HLA-DR1.01+ and 6 HLA-DR15.01+) compared to 24 HIV-uninfected HLA-matched subjects.

Conclusions/Significance

We demonstrated that MHC class II antigen processing and presentation is preserved in MN from HIV-infected individuals. This further supports the concept that this aspect of APC function does not further contribute to CD4+ T cell dysfunction in HIV disease.     

MRC OX-2 antigen: a lymphoid/neuronal membrane glycoprotein with a structure like a single immunoglobulin light chain.

The MRC OX-2 antigen is a rat cell surface glycoprotein of mol. wt. 41 000-47 000 found on neurones, thymocytes, B cells, follicular dendritic cells and endothelium. We now report the amino sequence for this antigen as deduced from the nucleotide sequence of cDNA clones detected by use of an oligonucleotide probe. The sequence contains 248 amino acid residues of which 202 residues are likely to be outside the cell with two domains that show homology with immunoglobulins. The N-terminal domain fits best with Ig V domains and Thy-1 antigen while the C-terminal part is like an Ig C domain. Thus the structure overall is similar to an Ig light chain or the T cell receptor beta chain. Three glycosylation sites are identified on each of the MRC OX-2 antigen domains.     

Studies on Miltenberger class III, V, Mv and Mk red cells. I. Sodium-dodecylsulfate polyacrylamide gel electrophoretic investigations

The glycoproteins in erythrocyte membrane from individuals exhibiting the rare alleles at the MNSs blood group locus Miltenberger (Mi-) III, V, Mv and Mk were studied by sodium-dodecylsulfate polyacrylamide gel electrophoretic techniques. The results suggest that the genes Mi-III and -V give rise to the formation of Ss sialoglycoproteins whose electrophoretic mobilities are altered. The Mi-V alteration is additionally associated with a decreased MN glycoprotein content. The allele Mv leads to a decreased Ss glycoprotein content. Data on ordinary Mk and Mk/Mi-III red cells suggest that the gene complex Mk does not give rise to the synthesis of Ss glycoprotein.     

One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes

Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47?kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.     

Bcl-2 enhances neurite extension via activation of c-Jun N-terminal kinase

Recent studies suggest that Bcl-2 may play an active role in neuronal differentiation. Here, we showed a marked neurite extension in MN9D dopaminergic neuronal cells overexpressing Bcl-2 (MN9D/Bcl-2) or Bcl-X(L) (MN9D/Bcl-X(L)). We found a specific increase in phosphorylation of c-Jun N-terminal kinase (JNK) accompanied by neurite extension in MN9D/Bcl-2 but not in MN9D/Bcl-X(L) cells. Consequently, neurite extension in MN9D/Bcl-2 but not in MN9D/Bcl-X(L) cells was suppressed by treatment with SP600125, a specific inhibitor of JNK. Inhibition of other mitogen-activated protein kinases-including p38 and extracellular signal-regulated kinase-did not affect Bcl-2-mediated neurite extension in MN9D cells. While the expression levels of such protein markers of maturation as SNAP-25, phosphorylated NF-H, and neuron-specific enolase were increased in MN9D/Bcl-2 cells, only upregulation of SNAP-25 was inhibited after treatment with SP600125. Thus, the JNK signal activated by Bcl-2 seems to play an important role during morphological and certain biochemical differentiation in cultured dopaminergic neurons.     

N-Terminal myristoylation predictions by ensembles of neural networks

N-terminal myristoylation is a post-translational modification that causes the addition of a myristate to a glycine in the N-terminal end of the amino acid chain. This work presents neural network (NN) models that learn to discriminate myristoylated and nonmyristoylated proteins. Ensembles of 25 NNs and decision trees were trained on 390 positive sequences and 327 negative sequences. Experiments showed that NN ensembles were more accurate than decision tree ensembles. Our NN predictor evaluated by the leave-one-out procedure, obtained a false positive error rate equal to 2.1%. That was better than the PROSITE pattern for myristoylation for which the false positive error rate was 22.3%. On a recent version of Swiss-Prot (41.2), the NN ensemble predicted 876 myristoylated proteins, while 1150 proteins were predicted by the PROSITE pattern for myristoylation. Finally, compared to the well-known NMT predictor, the NN predictor gave similar results. Our tool is available under http://www.expasy.org/tools/myristoylator/myristoylator.html.     

Differential processing of the neurotensin/neuromedin N precursor in the mouse

Posttranslational processing of the neurotensin/neuromedin N (NT/NN) precursor has been investigated in mouse brain and small intestine by means of region-specific radioimmunoassays coupled to chromatographic fractionations. In brain, total NT/NN immunoreactivity measured with a common C-terminal antiserum was 15.72 pmol/g. NT measured with an N-terminal antiserum was 9.74 pmol/g and NN measured with an N-terminal antiserum was 5.98 pmol/g. In small intestine, combined NT/NN immunoreactivity was 108.55 pmol/g, consisting of 66.37 pmol/g NT but only 0.96 pmol/g NN. Gel permeation chromatography and reverse phase HPLC revealed that the large discrepancy in the NT and NN values obtained in small intestinal extracts was due to the presence of a high molecular weight, hydrophobic peptide, which was reactive only with the common C-terminally directed antiserum. Pepsinization of this generated an immunoreactive peptide with similar chromatographic characteristics to NN. In mouse intestine, NN is only partially cleaved from the common NT/NN precursor, resulting in the presence of an N-terminally extended molecular species. This novel molecular species of neuromedin N may be the physiological mediator of certain peripheral biological effects hitherto attributed to neurotensin or neuromedin N.     

Concurrent enhancement of monocyte immunoregulatory properties and effector functions by recombinant interferon-gamma

Interferon-gamma is a critical factor in the activation of several mononuclear phagocyte effector and immunoregulatory properties. However, it remains uncertain if IFN-gamma is capable of concurrent activation of both functions in the same cell population. Plastic adherent mononuclear cells (80-98% MN by cytochemical criteria) were cultivated in the absence or presence of recombinant interferon-gamma (rIFN-gamma, 0.1-100 U/ml) for 48 hr. MN surface DR antigen was assessed by flow cytometry (EPICS V) after staining with monoclonal antibodies OKIa1 or L243. Exposure to rIFN-gamma (100 U/ml) increased MN surface DR antigen (mean fluorescence intensity) by 80 +/- 20% (P less than 0.01) and 121 +/- 52% (P less than 0.001), respectively, compared to untreated cells. The increase in DR antigen was maximal at 100 U/ml, dependent on protein and RNA synthesis and blocked by agents that increase cAMP levels. IL-1 activity was determined in the mouse thymocyte assay; rIFN-gamma (100 U/ml) increased IL-1 activity in the supernatants of MN cultured in medium alone from 0.5 +/- 0.2 to 7.8 +/- 4.7 U/ml (P less than 0.05), and lipopolysaccharide-stimulated MN from 20.4 +/- 19.1 to 71.7 +/- 38.9 U/ml (P less than 0.05). Following rIFN-gamma exposure, MN stimulation of the AMLR was increased at 6 days (29,269 +/- 5224 vs 13,252 +/- 4938 cpm, P less than 0.01). Spontaneous cytotoxicity (SC) and antibody-dependent cell cytotoxicity (ADCC) were studied in a 51Cr release microculture assay using the human lymphoblastoid cell line CCRF-CEM as target. SC by MN increased linearly as a function of log[rIFN-gamma] for effector:target (E:T) ratios of 5:1 (r = 0.95, P less than 0.01) and 10:1 (r = 0.99, P less than 0.01). ADCC by MN increased following rIFN-gamma exposure (100 U/ml) at E:T ratios of 5:1 (22 +/- 13 to 31 +/- 4%, P less than 0.025) and 10:1 (31 +/- 4 to 38 +/- 4%, P less than 0.01). Thus, rIFN-gamma not only activates MN effector function, but has concurrent stimulatory effects on multiple MN properties critical to immunoregulation.     

NMR dynamics and antibody recognition of the meningococcal lipidated outer membrane protein LP2086 in micellar solution

Neisseria meningitidis is a major cause of meningitis. Although protective vaccination is available against some pathogenic serogroups, serogroup B meningococci have been a challenge for vaccinologists. A family of outer membrane lipoproteins, LP2086 (or factor H binding proteins, fHbp), has been shown to elicit bactericidal antibodies and is currently part of a cocktail vaccine candidate. The NMR structure of the variant LP2086-B01 in micellar solution provided insights on the topology of this family of proteins on the biological membrane. Based on flow cytometry experiments on whole meningococcal cells, binding experiments with monoclonal antibodies, and the NMR structure in micellar solution, we previously proposed that LP2086-B01 anchors the outer bacterial membrane through its lipidated N-terminal cysteine, while a flexible 20 residue linker positions the protein above the layer of lipo-oligosaccharides that surrounds the bacteria. This topology was suggested to increase the antigen exposure to the immune system. In the present work, using micellar solution as a membrane mimicking system, we characterized the backbone dynamics of the variant LP2086-B01 in both its lipidated and unlipidated forms. In addition, binding experiments with a Fab fragment derived from the monoclonal MN86-1042-2 were also performed. Our data suggests that due to the length and flexibility of the N-terminal linker, the antigen is not in contact with the micelle, thus making both N- and C-domains highly available to the host immune system. This dynamic model, combined with the binding data obtained with MN86-1042-2, supports our previously proposed arrangement that LP2086-B01 exposes one face to the extracellular space. Binding of MN86-1042-2 antibody shows that the N-domain is the primary target of this monoclonal, providing further indication that this domain is immunologically important for this family of proteins.