Subunit-specific Monoclonal Antibodies Research Articles

P504 Guselkumab, an IL-23p19 subunit–specific monoclonal antibody, binds CD64+ myeloid cells and potently neutralises IL-23 produced from the same cells

Abstract Background Monoclonal antibodies targeting the interleukin (IL)-23p19 subunit are effective in the treatment of inflammatory bowel diseases (IBD), but have different molecular attributes that may translate to differences in clinical efficacy. Within this class, guselkumab (GUS) is a fully human IgG1 monoclonal antibody with a native Fc region, and risankizumab (RIS) is a humanised IgG1 antibody with a mutated Fc region. Binding of these therapeutic antibodies to Fcγ receptor (FcγR) I, or CD64, is of particular interest, as CD64+ IL-23-producing myeloid cells are increased in the inflamed colon in IBD and correlated with endoscopic disease severity (Chapuy L, et al. Mucosal Immunol. 2019; Chapuy et al. J Crohns Colitis. 2020). Here, we compared functional characteristics of the antigen-binding and Fc regions of GUS and RIS. Methods IL-23 binding affinity of GUS and RIS was compared in vitro using KinExA and surface plasmon resonance. In vitro cellular potency of GUS and RIS was measured by impact on IL-23-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in human peripheral blood mononuclear cells. Binding of GUS and RIS to FcγRs was assessed in cells transfected with individual FcγRs. In primary human “inflammatory” monocytes differentiated with granulocyte-macrophage colony-stimulating factor and interferon-γ (IFN-γ) followed by toll-like receptor stimulation to induce IL-23 production, binding of GUS and RIS to CD64 and capture of endogenously secreted IL-23 was assessed using flow cytometry. The potential impact of GUS binding to CD64 was assessed in IFN-γ primed monocytes using a human 41-plex cytokine bead assay. Results GUS and RIS displayed comparable picomolar binding affinity for IL-23 and equivalent high potency for inhibition of IL-23-induced STAT3 phosphorylation. GUS showed strongest binding to CD64 compared with other FcγRs, whereas RIS had negligible binding to any FcγR. GUS, but not RIS, showed dose-dependent Fc-mediated binding to CD64 in primary human “inflammatory” monocytes. Moreover, CD64-bound GUS was able to simultaneously capture IL-23 endogenously secreted from the same cells (Figure). GUS binding to CD64 on monocytes did not induce cytokine production. Conclusion GUS, but not RIS, can simultaneously bind CD64+ myeloid cells via its Fc region and neutralise IL-23 with high affinity and potency. Our in vitro data suggest a mechanistic benefit through enhanced localization of GUS within the inflamed colon, where CD64+ IL-23-producing myeloid cells are increased, and GUS can potently neutralise IL-23 at its source of production. These findings may contribute to differences in therapeutic profiles between antibodies.

MITOCHONDRIAL DISEASES (Posters): P.195A novel multiplex chromogenic immunoassay for evaluating mitochondrial respiratory chain complex I and complex IV defects in diagnostic muscle biopsies

The investigation of clinically suspected mitochondrial disease (mtD) includes performing a skeletal muscle biopsy for biochemical/histochemical assessment of mitochondrial respiratory chain (RC) defects. COX-SDH histochemistry detects RC-complex IV (CIV) defects, but RC-complex I (CI) defects cannot be detected histochemically. CI/CIV defects are common in mtD. Immunohistochemical evaluation of RC-complex defects relies on reduced amount of the assembled complex associated with catalytic deficiency, detectable with RC subunit-specific monoclonal antibodies. Our aim was to design a dual chromogenic immunoassay (DCI) for evaluating CI/CIV defects in diagnostic muscle biopsies. In the DCI optimised protocol, primary antibodies (Abcam), TOMM20 (mitochondrial mass), NDUFB8 (CI) and MTCO1 (CIV) were coincubated (TOMM20+CI and TOMM20+CIV), and then TOMM20 developed to yellow and the other marker to teal (Discovery/Ventana Systems) with colocalising antibodies visualising as green. Control sections stained as a mosaic dark green (type I fibres) and light green (type II fibres) pattern. Completely CI/CIV-deficient fibres stained yellow, and partly CI/CIV-deficient fibres stained yellow-green, and were easily detectable due to good visual colour contrast. The DCI and COX-SDH assays were performed in serial frozen sections. 23 biopsies were assessed: 15 with genetically confirmed mtD (mtDNA rearrangements/point mutations/depletion), 4 with high clinical/histological suspicion of mtD, and 4 unaffected controls. % COX and CI/CIV-deficient fibres were counted in two random fascicles, with high concordance amongst % COX-negative and CI/CIV-deficient fibres. The DCI detected more CI-deficient fibres in 7/19 cases and more CIV-deficient fibres in 5/19 cases compared to COX-negative fibres (average 6%). Most COX-negative fibres had dual CI+CIV defects with DCI. Segmental and partial CI/CIV defects were detectable. Equivocal COX-SDH stained fibres were often strongly CI/CIV-immunodeficient. In conclusion, our multiplex DCI reliably detects CI/CIV defects comparable in sensitivity to the COX-SDH histochemical assay, is easy to evaluate due to a good visual contrast between CI/CIV positive and negative fibres and can be easily co-opted to routine diagnostic work. Studies are underway to develop a quadruple chromogenic immunoassay for digital evaluation of CI/CIV defects.

Distinctive expression of interleukin-23 receptor subunits on human Th17 and γδ T cells.

The interleukin-23 (IL-23) pathway, T helper 17 (Th17) cells and γδ T cells, which respond to IL-23, have major pro-inflammatory roles. We have used unique IL-23 receptor (IL-23R) subunit-specific monoclonal antibodies, X67 and X68, and IL-12 receptor beta-1 subunit (IL-12Rβ1) expression levels to evaluate the IL-23R complex on CD4 αβ TCR Th17 cells and on γδ T cells. Both IL-23R and IL-12Rβ1 subunits constitute the functional IL-23R. Expression of the IL-23R subunit by cultured Th17 cells was heterogeneous. Th17 cells expressed consistent high levels of the IL-12Rβ1 subunit, which appeared a better predictor of responsiveness to IL-23 than the expression of the IL-23R subunit. Moreover, sorting memory CD4 T cells by high IL-12Rβ1 expression selectively enriched cells committed to IL-17 production from the blood. IL-23R expression was also observed on freshly isolated and cultured γδ T cells and the cultured γδ T cells were not responsive to IL-23.

Allosteric property of the (Na ++K +)-ATPase β 1 subunit

(Na ++K +)-ATPase (NKA) comprises two basic α and β subunits: The larger α subunit catalyzes the hydrolysis of ATP for active transport of Na + and K + ions across the plasma membrane; the smaller β subunit does not take part in the catalytic process of the enzyme. Little is known about allosteric regulation of the NKA β subunit. Here, we report a surprising finding that extracellular stimuli on the native β 1 subunit can generate a significant impact on the catalytic function of NKA. By using a β 1 subunit-specific monoclonal antibody JY2948, we found that the JY2948–β 1 subunit interaction markedly enhances the catalytic activity of the enzyme and increases the apparent affinity of Na + and K + ions for both ouabain-resistant rat NKA and ouabain-sensitive dog NKA. This study provides the first evidence to identify an allosteric binding site residing on the NKA β 1 subunit and uncovers the latent allosteric property of the β 1 subunit, which remotely controls the NKA catalytic function.

Immunofluorometric Point-of-Care Assays for the Detection of Acute Coronary Syndrome-Related Noncomplexed Pregnancy-Associated Plasma Protein A

We recently reported that the pregnancy-associated plasma protein A (PAPP-A) form specifically related to acute coronary syndromes (ACS) is not complexed with the proform of eosinophil major basic protein (proMBP). The aim of this study was to develop rapid point-of-care immunoassays for the measurement of the noncomplexed PAPP-A. We developed immunofluorometric noncompetitive dry-reagent assays for total PAPP-A with 2 PAPP-A subunit-specific monoclonal antibodies and for PAPP-A/proMBP complex with 1 PAPP-A subunit-specific antibody and 1 proMBP subunit-specific antibody. The concentration of noncomplexed PAPP-A was determined as the difference of the results obtained with the 2 assays. The assays were linear from 0.5 to 300 mIU/L. The analytical detection limit and functional detection limit (CV <20%) were 0.18 mIU/L and 0.27 mIU/L for total PAPP-A assay and 0.23 mIU/L and 0.70 mIU/L for PAPP-A/proMBP assay, respectively. The total assay imprecisions were <10%, and recoveries were 88%-107% for both assays. The mean difference (95% limits of agreement) between the new total PAPP-A assay and a previously reported total PAPP-A assay was -3.2% (-45.7% to 39.3%; n = 546; P = 0.0019). In serum samples from 159 non-ACS individuals, median concentrations (interquartile range) were 2.42 (1.14) mIU/L for total PAPP-A, 2.20 (1.18) mIU/L for PAPP-A/proMBP, and 0.18 (0.63) mIU/L for noncomplexed PAPP-A. Total PAPP-A and PAPP-A/proMBP, but not noncomplexed PAPP-A, correlated with age (r = 0.290, P = 0.0002; r = 0.230, P = 0.0035; r = 0.075, P = 0.3483, respectively). The new assays described revealed that noncomplexed PAPP-A is found only in negligible amounts in non-ACS samples.

Development and characterization of human constitutive proteasome and immunoproteasome subunit‐specific monoclonal antibodies

Delta (Y), MB1 (X), and Z are the three catalytic beta-subunits located in the inner rings of the constitutive proteasome, an intracellular multicatalytic complex responsible for the generation of peptides presented by human leukocyte antigen (HLA) class I antigens to T cells. When cells are incubated with interferon-gamma, delta (Y), MB1 (X), and Z are replaced by LMP2, LMP7, and LMP10, respectively, leading to the expression of immunoproteasome which generates peptides with increased affinity for HLA class I antigens. The characterization of the expression of constitutive proteasome and immunoproteasome subunits in cells, normal tissues, and malignant lesions has been hampered by the lack or limited availability of constitutive proteasome and immunoproteasome subunit-specific monoclonal antibodies (mAbs), which are suitable for immunohistochemical staining. To overcome this limitation, we generated human delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10-specific mAb-secreting hybridomas from BALB/c mice immunized with peptides and recombinant fusion proteins. The mAbs SY-5, SJJ-3, NB-1, SY-1, HB-2, and TO-7 were shown to be specific for delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10, respectively, as they react specifically with the corresponding molecules when tested with a human B lymphoid LG2 cell lysate in Western blotting and with the peptide derived from each molecule in enzyme-linked immunosorbent assay. The reactivity of the six mAbs with the corresponding intracellular antigens resulted in intracellular staining when the mAbs were tested with microwave-treated and saponin-permeabilized cells in indirect immunofluorescence and with formalin-fixed, paraffin-embedded tissue sections in immunohistochemical reactions. These results suggest that the constitutive proteasome and immunoproteasome subunit-specific mAbs we have developed are useful probes to characterize the expression of proteasome subunits in normal tissues and in pathological lesions.

Cellular and synaptic distribution of NR2A and NR2B in macaque monkey and rat hippocampus as visualized with subunit-specific monoclonal antibodies

The functional and pharmacological attributes of the N-methyl- d-aspartate (NMDA) receptor are related to its subunit composition, thus resolving the subunit composition of NMDA receptors in specific classes of synapses is an important step in characterizing excitatory circuits. Toward this end, mouse monoclonal antibodies were raised against fusion protein antigens corresponding to the putative amino acid sequences of human NMDA receptor subunits NR2A and NR2B. The subunit specificity of these monoclonal antibodies was demonstrated with transfected human and rat NMDA receptor cDNAs, and their immunoreactivity was established in rat, macaque monkey, and human brain tissue. At the light microscopic level, both NR2A and NR2B exhibit a distribution in monkey and rat hippocampus very similar to NMDA receptor subunit NR1, and both are highly colocalized with NR1. Electron microscopic immunogold studies demonstrated that both NR2A and NR2B are often present in asymmetric synapses in CA1, commonly colocalized with NR1, and often colocalized with each other in the same asymmetric synapses. Both assembly and synthetic pools are present within spines and spine necks, respectively, particularly for NR2A. The confocal and ultrastructural data suggest that whereas NR1, NR2A, and NR2B are essentially uniformly colocalized in hippocampal projection neurons, there is extensive heterogeneity at the synaptic level that would lead to multiple functional classes of NMDA receptor-mediated synapses, and extensive capacity for plasticity at the synapse. Thus, the subunit profile of a given synapse may be dynamic, with regulation of local synthesis and insertion of different subunits into the synapse leading to a complex, heterogeneous, and shifting set of functional attributes of the NMDA receptor.

Enzyme-linked immunosorbent assay of total inhibin: direct determination based on inhibin α subunit-specific monoclonal antibodies

Objective: Inhibin circulates in various molecular weight forms. Alpha (α)-subunit-directed total inhibin immunoassays, which detect all forms of α subunits plus the α/β inhibin dimers, have been found valuable in the diagnosis and monitoring of ovarian cancer. Because of the dependency of the published methods on boiling sample pre-treatment with SDS and unavailability of a commercial assay, we developed an enzyme-linked immunosorbent assay (ELISA) for direct determination of total inhibin. Design and methods: Method development involved a pair of well-characterized inhibin α subunit-directed antibodies and determination of the effects of various assay parameters. Selection of the optimized protocol was guided by the outcome of comparative sample analysis using previously reported boiling sample pre-treatment reagents and protocols. Results: We report development of a simplified ELISA for total inhibin. Method evaluation data demonstrated acceptable analytical performance characteristics with detection limit of 2 ng/l (recombinant inhibin-A), dynamic range of 12.5–500 ng/l, and intra- and inter-assay imprecision of 2.3–4.6% and 3.3–5.1% at total inhibin concentrations of approximately 60–400 ng/l, respectively. The mean (±SD) recovery from spiked serum samples averaged 109 ± 14% and recovery in response to serial sample dilution was 99 ± 10%. Serum values by the direct method ( n = 40) correlated strongly with those obtained after sample pre-treatment by boiling with SDS ( r = 0.97). As expected, the total inhibin immunoreactivity in human follicular fluid fractionated by HPLC gel filtration in multiple immunoreactive peaks (8–250 kDa). In serum samples from postmenopausal women with ovarian cancer, the assay detected significantly higher total inhibin levels than in samples from normal postmenopausal controls. Conclusion: The development of a fast and simplified ELISA should facilitate wider investigations of pathophysiology and diagnostic potential of total inhibin measurement.

Immunohistochemical localization of sodium-potassium ATPase in human normal stomach and gastric adenocarcinomas.

We studied the expression pattern of Na, K-ATPase beta 1 subunit in human normal stomachs and in gastric adenocarcinomas by using anti-Na, K-ATPase beta 1 subunit-specific monoclonal antibody. Tissue samples were processed in formalin solution or in a cold acetic acid-ethanol solution, routinely processed, embedded in paraffin, and an immunoperoxidase method for Na, K-ATPase beta 1 subunit was performed. After antigen retrieval using a steamer in citrate buffer (pH 6.0), tissue sections initially fixed in cold acetic acid-ethanol showed intense immunoreactivity with the antibody at the lateral or basolateral cytoplasmic membrane of normal gastric epithelial cells, at the cytoplasmic membrane of gastric carcinoma cells according to the level of differentiation, and at the cytoplasmic membrane and in the cytoplasm of Schwann cells and neurons in the mesenteric plexus of the gastric wall. Acetic acid-ethanol and paraffin embedding is a useful method for the investigation of the immunohistochemical localization of Na, K-ATPase in normal and diseased tissues.

Assembly of the Drosophila 26 S proteasome is accompanied by extensive subunit rearrangements.

The subunit contacts in the regulatory complex of the Drosophila 26 S proteasome were studied through the cross-linking of closely spaced subunits of the complex, and analysis of the cross-linking pattern in an immunoblot assay with the use of subunit-specific monoclonal antibodies. The cross-linking pattern of the purified 26 S proteasome exhibits significant differences as compared with that of the purified free regulatory complex. It is shown that the observed differences are due to extensive rearrangement of the subunit contacts accompanying the assembly of the 26 S proteasome from the regulatory complex and the 20 S proteasome. Cross-linking studies and electron microscopic examinations revealed that these changes are reversible and follow the assembly or the disassembly of the 26 S proteasome. Although the majority of the changes observed in the subunit contacts affected the hexameric ring of the ATPase subunits, the alterations extended over the whole of the regulatory complex, affecting subunit contacts even in the lid subcomplex. Changes in the subunit contacts, similar to those in the regulatory complex, were detected in the 20 S proteasome. These observations indicate that the assembly of the 26 S proteasome is not simply a passive docking of two rigid subcomplexes. In the course of the assembly, the interacting subcomplexes mutually rearrange their structures so as to create the optimal conformation required for the assembly and the proper functioning of the 26 S proteasome.

Targeting α7-containing nicotinic receptors on neurons to distal locations

Nicotinic receptors containing the α7 gene product are widely expressed in the nervous system and have a high relative permeability to Ca 2+. This permits them to influence a variety of Ca 2+-dependent events in neurons. On chick ciliary ganglion neurons, the receptors are concentrated on somatic spines and contribute directly to postsynaptic signaling. Receptors containing the α7 gene product can also be found in the chick sciatic nerve being transported to distal locations. Both motoneurons and dorsal root ganglion neurons are candidate sources of the receptors since both extend processes into the nerve and synthesize α7 protein. Immunoprecipitation assays with subunit-specific monoclonal antibodies and pharmacological comparisons fail to detect differences between sciatic nerve and ciliary ganglion α7-containing receptors. Cell-specific machinery and receptor posttranslational modifications may determine which sites the receptors populate.

A Missense Mutation of Cytochrome Oxidase Subunit II Causes Defective Assembly and Myopathy

We report the first missense mutation in the mtDNA gene for subunit II of cytochrome c oxidase (COX). The mutation was identified in a 14-year-old boy with a proximal myopathy and lactic acidosis. Muscle histochemistry and mitochondrial respiratory-chain enzymology demonstrated a marked reduction in COX activity. Immunohistochemistry and immunoblot analyses with COX subunit-specific monoclonal antibodies showed a pattern suggestive of a primary mtDNA defect, most likely involving CO II, for COX subunit II (COX II). mtDNA-sequence analysis demonstrated a novel heteroplasmic T-->A transversion at nucleotide position 7,671 in CO II. This mutation changes a methionine to a lysine residue in the middle of the first N-terminal membrane-spanning region of COX II. The immunoblot studies demonstrated a severe reduction in cross-reactivity, not only for COX II but also for the mtDNA-encoded subunit COX III and for nuclear-encoded subunits Vb, VIa, VIb, and VIc. Steady-state levels of the mtDNA-encoded subunit COX I showed a mild reduction, but spectrophotometric analysis revealed a dramatic decrease in COX I-associated heme a3 levels. These observations suggest that, in the COX protein, a structural association of COX II with COX I is necessary to stabilize the binding of heme a3 to COX I.

Biochemical, genetic and immunoblot analyses of 17 patients with an isolated cytochrome c oxidase deficiency

Mitochondrial respiratory chain defects involving cytochrome c oxidase (COX) are found in a clinically heterogeneous group of diseases, yet the molecular basis of these disorders have been determined in only a limited number of cases. Here, we report the clinical, biochemical and molecular findings in 17 patients who all had isolated COX deficiency and expressed the defect in cultured skin fibroblasts. Immunoblot analysis of mitochondrial fractions with nine subunit specific monoclonal antibodies revealed that in most patients, including in a patient with a novel mutation in the SURF1 gene, steady-state levels of all investigated COX subunits were decreased. Distinct subunit expression patterns were found, however, in different patients. The severity of the enzymatic defect matched the decrease in immunoreactive material in these patients, suggesting that the remnant enzyme activity reflects the amount of remaining holo-enzyme. Four patients presented with a clear defect of COX activity but had near normal levels of COX subunits. An increased affinity for cytochrome c was observed in one of these patients. Our findings indicate a genetic heterogeneity of COX deficiencies and are suggestive of a prominent involvement of nuclear genes acting on the assembly and maintenance of cytochrome c oxidase.

Functional Implications of the Subunit Composition of Neuronal CaM Kinase II

The assembly of 6-12 subunits of Ca(2+)/calmodulin-dependent kinase II (CaM kinase II) into holoenzymes is an important structural feature of the enzyme and its postulated role as a molecular detector of Ca(2+) oscillations. Using single cell reverse transcriptase-polymerase chain reaction, we show that alpha- and beta-CaM kinase II mRNAs are simultaneously present in the majority of hippocampal neurons examined and that co-assembly of their protein products into heteromers is therefore possible. The subunit composition of CaM kinase II holoenzymes was analyzed by immunoprecipitation with subunit-specific monoclonal antibodies. Rat forebrain CaM kinase II consists of heteromers composed of alpha and beta subunits at a ratio of 2:1 and homomers composed of only alpha subunits. We examined the functional effect of the heteromeric assembly by analyzing the calmodulin dependence of autophosphorylation. Recombinant homomers of alpha- or beta-CaM kinase II, as well as of alternatively spliced beta isoforms, have distinct calmodulin dependences for autophosphorylation based on differences in their calmodulin affinities. Half-maximal autophosphorylation of alpha is achieved at 130 nM calmodulin, while that for beta occurs at 15 nM calmodulin. In CaM kinase II isolated from rat forebrain, however, the calmodulin dependence for autophosphorylation of the beta subunits is shifted toward that of alpha homomers. This suggests that Thr(287) in beta subunits is phosphorylated by alpha subunits present in the same holoenzyme. Once autophosphorylated, beta-CaM kinase II traps calmodulin by reducing the rate of calmodulin dissociation.

Epitope mapping of a monoclonal antibody directed against the alpha-subunit of phosphofructokinase-1 from Saccharomyces cerevisiae by screening phage display libraries.

Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of two types of subunits, alpha and beta. Subunit-specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, alpha-F3, binds to an epitope either at the C-terminal or at the N-terminal part of the alpha-polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D-A-F and D-S-F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody alpha-F3 to native phosphofructokinase-1. The peptide G-I-K-D-A-F-L inhibited the binding more strongly (IC50 = 1.5 microM) than the peptide A-P-W-H-D-S-F (IC50 = 33.3 microM). Sequence matching revealed the presence of the D-A-F motif in the polypeptide chain of phosphofructokinase-1 at amino acid position 172-174. As a control, the nonapeptide A-P-T-S-K-D-A-F-L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC50 = 0.3 microM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase-1 that is recognized by antibody alpha-F3.

Nicotinic receptor subtypes in the developing chick brain: appearance of a species containing the alpha4, beta2, and alpha5 gene products.

Increasing evidence suggests nicotinic receptors regulate developmental events in the nervous system. We used [3H]epibatidine and 125I-alpha-bungarotoxin, together with subunit-specific monoclonal antibodies, to distinguish and quantify nicotinic receptor subtypes in developing chick brain. The results show that more than three fourths of the epibatidine-binding receptors at both early and late embryonic stages contain alpha4 and beta2 subunits, representing receptors previously distinguished by high affinity nicotine binding. A fraction of these also contain the alpha5 gene product, which is consistent with studies on transfected cells showing that the alpha4, beta2, and alpha5 gene products coassemble to produce epibatidine-binding receptors. A small portion of the receptors contain alpha3 and beta4 subunits, assembled in part with either alpha4 or beta2 subunits. The most abundant nicotinic receptors, however, at both early and late embryonic stages are those having high affinity for alpha-bungarotoxin rather than epibatidine. Most contain alpha7 subunits, whereas about half contain alpha8 subunits as well. The sharpest developmental increase between embryonic days 8 and 17/18 occurs with receptors containing alpha5 subunits, whereas receptors containing alpha3 or beta4 subunits undergo no specific increase. The three major receptor species (containing alpha4 and beta2 but not alpha5 subunits; alpha7 subunits; or alpha7 and alpha8 subunits) each increase approximately 3-fold during the same period. The results indicate greater receptor complexity than appreciated previously; they provide information about the rules governing subunit assembly in neuronal nicotinic receptors and draw attention to the role of alpha5 subunits in late development.

Mutants of human choriogonadotropin lacking N-glycosyl chains in the alpha-subunit. 1. Mechanism for the differential action of the N-linked carbohydrates.

Analogs of human choriogonadotropin (hCG) lacking N-glycosyl chains at alpha52Asn and alpha78Asn were purified from the culture media of insect cells by immunoaffinity chromatography using a monoclonal antibody column. As previously reported, while analogs lacking carbohydrate at alpha52Asn and alpha78Asn had similar receptor binding activities compared with the wild type recombinant hCG (hCGwt), they differed in their signal transduction properties. The mutant lacking carbohydrate at alpha78Asn had 20% less cAMP-stimulating activity than hCGwt, but the absence of glycosylation at alpha52Asn resulted in the reduction of cAMP accumulation by 90-95%. A similar effect of the mutations was observed on the stimulation of steroidogenesis. Circular dichroism spectra of the two mutants showed significant differences. The mutant lacking carbohydrate at alpha52Asn had a much higher negative mean residue ellipticity (MRE) at 200 nm and a lower negative MRE at 220 nm than that lacking carbohydrate at alpha78Asn and hCGwt. The dissociation rates of the alpha52Asn and alpha78Asn carbohydrate deficient mutants at pH 3 and room temperature, measured by using 1-anilino-8-naphthalenesulfonate, were 9.4 x 10(-5) and 3.8 x 10(-5) s-1, respectively, as compared with 1.5 x 10(-5) s-1 for hCGwt. The results of both CD measurements and dissociation studies strongly suggest that the absence of carbohydrate at alpha52Asn results in conformational changes in the mutant which might explain the loss in its signal transduction function. This is further supported by indirect evidence from two other lines of experimentation. Unlike the mutant lacking carbohydrate at alpha78Asn, the one lacking carbohydrate at alpha52Asn cross-reacted with the two subunit specific monoclonal antibodies, anti-hCGalpha and anti-hCGbeta, which normally did not cross-react with the native or the hCGwt. Also, polyclonal anti-hCGbeta but not anti-hCGalpha was able to restore the cAMP-producing activity of the alpha52Asn carbohydrate deficient mutant. From all the data taken together, it appears that the loss of second messenger-producing activity of hCG with the absence of the glycosyl chain at alpha52Asn was probably due to a conformational change in the heterodimer rather than due to the loss of the alpha52Asn-carbohydrate-receptor interaction.

Inhibition of integrin mediated cell adhesion of human head and neck squamous cell carcinoma to extracellular matrix laminin by monoclonal antibodies

We recently reported that three members of the integrin family of cell adhesion molecules, designated alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4, are expressed at increased levels within the tumors and cell lines of patients with SCC. These three integrins have been reported to serve as receptors for laminin isoforms, and we also previously observed that laminins are secreted by SCC cell lines isolated from patients. In this study, the expression and localization of the three integrins and laminin in situ was evaluated in ten tumor specimens from patients with SCC by immunohistochemistry using integrin subunit-specific monoclonal antibodies. The ability of the antibodies to inhibit laminin attachment of a human squamous cell carcinoma line was determined by in vitro cell adhesion assay. Laminin and the three integrins were co-localized along the invasive border of the tumor parenchyma in 10/10 patient tumor specimens. Attachment of the UM-SCC-38 cell line to laminin was strongly inhibited by specific mAbs to alpha 2 and alpha 6 integrin subunits alone, or completely using a combination of alpha 2, alpha 3, and alpha 6 subunit specific mAbs. The co-localization of the three abnormally expressed integrins and laminin in patient tumor specimens indicates the potential for interaction of these receptors and ligand in vivo. The results of the cell adhesion assays using a patient SCC cell line that expresses the same repertoire of integrins confirms that SCC attach to laminin isoforms primarily through the alpha 2, alpha 3 and alpha 6 subunit-containing integrins. These findings provide a basis for undertaking experimental studies to obtain small molecule receptor antagonists to determine the role of these integrins in tumor formation, growth, invasion and metastasis in vivo.

Detection of antibody classes and subpopulations in myasthenia gravis patients using a new nonradioactive enzyme immunoassay.

To investigate the presence of antibodies in myasthenia gravis (MG) patients, we have developed a new reproducible and sensitive enzyme immunoassay (EIA-AChR), in which a beta subunit-specific monoclonal antibody (mAb 73) immobilizes fetal calf acetylcholine receptors (AChRs). We tested 92 MG patients (42 with positive and 50 with negative antibody titers), 60 healthy controls, and 40 controls with other autoimmune diseases. EIA-AChR detected immunoglobulin G (IgG) titers in all of the seropositive samples, with a significant correlation between these and those obtained using the traditional immunoprecipitation method. Moreover, 5 seronegative patients at immunoprecipitation assay were positive at EIA-AChR. EIA-AChR was also useful in revealing: (1) a seropositive patient subpopulation with generalized MG who had Abs directed against alpha-Bungarotoxin binding sites; and (2) patients with IgM directed against fetal calf AChR (detected in 13 seronegative and 16 seropositive MG patients, and in 6 of the patients with other autoimmune diseases).

Expression of the nicotinic receptor alpha 7 gene in tendon and periosteum during early development.

One of the most abundant nicotinic acetylcholine receptors expressed in the central and peripheral nervous systems is a species that contains the alpha 7 gene product, binds alpha-bungarotoxin with high affinity, and has a high relative permeability to calcium. The alpha 7 gene is also expressed at low levels in embryonic muscle tissue. We show here that the alpha 7 gene is expressed in tendon fibroblasts and periosteal cells during development. In situ hybridizations identify alpha 7 transcripts in tissue sections containing embryonic tendon and periosteum. RNase protection experiments demonstrate alpha 7 mRNA in primary tendon cells grown in culture. Immunofluorescence with subunit-specific monoclonal antibodies reveals alpha 7 protein in embryonic tendon. Immunoprecipitation assays with the antibodies indicate that the alpha 7-containing species in tendon is capable of binding alpha-bungarotoxin and that a similar species can be identified at low levels on the surface of fibroblasts in culture. The results show that the alpha 7 gene product is expressed in a range of tissues, including cells thought to be nonexcitable. The distribution of alpha 7 expression early in development and the ability of alpha 7-containing receptors to elevate intracellular calcium suggest that the gene may influence a variety of calcium-dependent events during embryogenesis.