Modulation of the activity of the transverse tubule Mg2+ATPase from frog skeletal muscle by a monoclonal antibody in vitro

We have established several hybridoma lines that produce monoclonal antibodies against transverse tubule (t-tubule) proteins from frog skeletal muscle. The specificity of these antibodies was characterized by ELISA and Western immunoblotting with purified t-tubule, sarcoplasmic reticulum and partially purified sarcolemmal membranes. One of the monoclonal antibodies (2/34.4) recognizes a band of 109 000 Da in immunoblots. When purified frog t-tubule vesicles were preincubated with this antibody we observed an increase in the rate of the Mg²⁺-ATPase enzyme (up to six fold) which was dependent on antibody concentration. Immunocytological experiments done on cryostat sections of frog muscle indicate that the antigen recognized by this antibody is localized mainly at the level of the t-tubules (I band) and to a lesser extent at the sarcolemma. These results indicate that monoclonal antibody 2/34.4 recognizes the t-tubule Mg²⁺-ATPase and modulates its activity. This antibody should be useful as a probe on studies designed to study the physiological function of the enzyme.Abbreviations t-tubules transverse-tubules - mAb monoclonal antibody - SR sarcoplasmic reticulum - SL sarcolemma     

Characterization of the major sulfated protein of mouse pancreatic acinar cells: A high molecular weight peripheral membrane glycoprotein of zymogen granules

The major sulfated protein of the mouse pancreatic acinar cell, gp300, hsa been identified and characterized with monoclonal and polyclonal antibidies. gp300 is a glycoprotein of M_r = 300,000 which contains ～40% of metabolically incroporated [³⁵S] sulfate in the acinar cell. Sulfate on gp300 is resistant to hot 1N HCl, but sensitive to alkaline hydrolysis. demonstrating that the sulfate is carbohydrate-linked rather than tyrosine-linked. gp300 metabolically labeled with [^3H]glucosamine and [³⁵H]sulfate was chemically and enzymaticlly treated followed by Bio-Gel P-10 gel filtration. Both labels were resistant to treatments which degrade glycosaminoglycan. Treatment of dual-labeled gp300 with PNGase F to cleave N-linked oligosaccharides released ～17% of [₃S]. Mild alkaline borohydride treatment after removal of N-linked sugar relased the remainder of both labels, indicating the presence of sulfated O-linked oligosaccharides. Biosynthetic studies and PNGase F digestion indicating the presence of sulfated O-linked oligosaccharides. Biosunynthetic studies and PNGase digestion F digestion indicate that the core protein is ～210 KDa, with apparent contrinution of ～35 KDa N-linked sugar, and ～55 KDa O-linked sugar. Lectin blotting and glycosidase digestion demonstrated the presece of Galβ(1–3)GalNAc and sialic acid α(2–3)Gal in O-linked oligosaccharide, and Galβ(1–4)GLcNAc in N-linked oligosaccharide. Immunolocalization and subcellular fractionation showed that gp300 is a peripheral memberane protein localized to the lumenal face of the zymogen granule membrane. gp300 was not secreted in reponse to hormone stimulation ofacini, so it is not a secertroy product. Immunoblot analysis showed that gp300 is present in other gastrointestinal tissues and parotid glands. Localization of this nonsecreted sulfated glycoprotein to exocrine secretory granule membranes suggests that gp300 may have a role in granule bigeneses.     

Structure of the N-linked oligosaccharide of the main diagnostic antigen of the pathogenic fungus Paracoccidiodes brasiliensis

The major diagnostic antigen of Paracoccidioides brasiliensisis the exocellularly secreted 43,000 Da glycoprotein (gp43)which contains a single N-linked oligosaccharide chain. Thisoligosaccharide, although poorly immunogenic in man, is responsiblefor the cross-reactivity of the gp43 with sera from patientswith histoplasmosis, and may have a role in fungal virulence.It contains a neutral highmannose core (Man₇GlcNAc₂) to whicha (1     

The genotype of early-transmitting HIV gp120s promotes α (4) β(7)-reactivity, revealing α (4) β(7) +/CD4+ T cells as key targets in mucosal transmission

Mucosal transmission of HIV is inefficient. The virus must breach physical barriers before it infects mucosal CD4⁺ T cells. Low-level viral replication occurs initially in mucosal CD4⁺ T cells, but within days high-level replication occurs in Peyer''s patches, the gut lamina propria and mesenteric lymph nodes. Understanding the early events in HIV transmission may provide valuable information relevant to the development of an HIV vaccine. The viral quasispecies in a donor contracts through a genetic bottleneck in the recipient, such that, in low-risk settings, infection is frequently established by a single founder virus. Early-transmitting viruses in subtypes A and C mucosal transmission tend to encode gp120s with reduced numbers of N-linked glycosylation sites at specific positions throughout the V1-V4 domains, relative to typical chronically replicating isolates in the donor quasispecies. The transmission advantage gained by the absence of these N-linked glycosylation sites is unknown. Using primary α₄β₇ ⁺/CD4⁺ T cells and a flow-cytometry based steady-state binding assay we show that the removal of transmission-associated N-linked glycosylation sites results in large increases in the specific reactivity of gp120 for integrin- α₄β₇. High-affinity for integrin α₄β₇, although not found in many gp120s, was observed in early-transmitting gp120s that we analyzed. Increased α₄β₇ affinity is mediated by sequences encoded in gp120 V1/V2. α₄β₇-reactivity was also influenced by N-linked glycosylation sites located in C3/V4. These results suggest that the genetic bottleneck that occurs after transmission may frequently involve a relative requirement for the productive infection of α₄β₇ ⁺/CD4⁺ T cells. Early-transmitting gp120s were further distinguished by their dependence on avidity-effects to interact with CD4, suggesting that these gp120s bear unusual structural features not present in many well-characterized gp120s derived from chronically replicating viruses. Understanding the structural features that characterize early-transmitting gp120s may aid in the design of an effective gp120-based subunit vaccine.     

Purification and properties of a highly active catalase from cabbage loopers,Trichoplusia ni

Using ethanol-chloroform fractionation in conjunction with standard column chromatography techniques catalase has been purified to electrophoretic homogeneity from mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni. The specific activity of purified catalase was 2.2 × 10⁵ units (IU = 1 μmol H₂O₂ decomposed mg protein⁻¹ min⁻¹). The purified enzyme's native molecular weight was in the 247,000–259,000 Da range and was tetrameric with an apparent molecular weight of 63,000 Da for each subunit. In addition, biochemical properties of the enzyme were studied with emphasis on substrate specificity, kinetics, and the mechanism of inactivation by the irreversible inhibitor 3-amino-1,2,4-triazole (AT). The apparent K_m of the purified catalase for H₂O₂ was 54.2 mM and 50% of the maximal rate occurred at 16 mM H₂O₂. Purified catalase was ineffective in metabolizing organic hydroperoxides and, unlike other catalases, lacked peroxidase activity. Lastly, AT in the presence and absence of H₂O₂ was an effective inhibitor of catalase activity (I₅₀ = 100 mM) suggesting that a portion of the purified catalase was complexed with hydrogen peroxide in a compound 1 configuration.     

The 43-kDa glycoprotein from the human pathogen Paracoccidioides brasiliensis and its deglycosylated form: excretion and susceptibility to proteolysis

Biochemical properties of the concanavalin A-binding 43-kDa glycoprotein (gp43) of Paracoccidioides brasiliensis and its deglycosylated form were compared. Deglycosylation was achieved by treatment with trifluoromethanesulfonic acid, endoglycosidase H, N-glycanase, or metabolically, by growing cells with tunicamycin. The resulting antigen in all cases had Mr 38,000, and probably derived from the gp43 by loss of N-linked high-mannose oligosaccharide chains. The presence of galactopyranose units in the carbohydrate chains was suggested by antigen binding to peanut lectin. Pulse and chase experiments using [35S]methionine metabolic labeling of P. brasiliensis growing in the presence of tunicamycin showed that the N-linked chains of gp43 are not required for antigen secretion. The 38-kDa antigen was more susceptible than the native antigen to the action of papain and pronase, thus indicating a protective role of the carbohydrate moiety against proteolysis. Both forms are equally resistant to endogenous proteases at neutral pH. The gp43, itself, has a proteolytic activity at pH 5-6, but not at neutral pH. Deglycosylation with endoglycosidase H or tunicamycin preserved epitopes in the 38-kDa molecule reactive with (a) antibodies from patients with paracoccidioidomycosis, or rabbit immunized with the gp43 and (b) mouse monoclonal antibodies against the gp43 antigen. The present results provide a basis for the understanding of diagnostic reactions and fungal virulence involving the gp43 exocellular antigen of P. brasiliensis.     

Differential reactivity of germ line allelic variants of a broadly neutralizing HIV-1 antibody to a gp41 fusion intermediate conformation

Genetic factors, as well as antigenic stimuli, can influence antibody repertoire formation. Moreover, the affinity of antigen for unmutated naïve B cell receptors determines the threshold for activation of germinal center antibody responses. The gp41 2F5 broadly neutralizing antibody (bNAb) uses the V_H2-5 gene, which has 10 distinct alleles that use either a heavy-chain complementarity-determining region 2 (HCDR2) aspartic acid (D_H54) or an HCDR2 asparagine (N_H54) residue. The 2F5 HCDR2 D_H54 residue has been shown to form a salt bridge with gp41 ⁶⁶⁵K; the V_H2-5 germ line allele variant containing N_H54 cannot do so and thus should bind less avidly to gp41. Thus, the induction of 2F5 bNAb is dependent on both genetic and structural factors that could affect antigen affinity of unmutated naïve B cell receptors. Here, we studied allelic variants of the V_H2-5 inferred germ line forms of the HIV-1 gp41 bNAb 2F5 for their antigen binding affinities to gp41 linear peptide and conformational protein antigens. Both V_H2-5 2F5 inferred germ line variants bound to gp41 peptides and protein, including the fusion intermediate protein mimic, although more weakly than the mature 2F5 antibody. As predicted, the affinity of the N_H54 variant for fusion-intermediate conformation was an order of magnitude lower than that of the D_H54 V_H2-5 germ line antibody, demonstrating that allelic variants of 2F5 germ line antibodies differentially bind to gp41. Thus, these data demonstrate a genetically determined trait that may affect host responses to HIV-1 envelope epitopes recognized by broadly neutralizing antibodies and has implications for unmutated ancestor-based immunogen design.     

Emergence of gp120 V3 Variants Confers Neutralization Resistance in an R5 Simian-Human Immunodeficiency Virus-Infected Macaque Elite Neutralizer That Targets the N332 Glycan of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

Neutralization-resistant simian-human immunodeficiency virus AD8 (SHIV_AD8) variants that emerged in an infected macaque elite neutralizer targeting the human immunodeficiency virus type 1 (HIV-1) gp120 N332 glycan acquired substitutions of critical amino acids in the V3 region rather than losing the N332 glycosylation site. One of these resistant variants, carrying the full complement of gp120 V3 changes, was also resistant to the potent anti-HIV-1 monoclonal neutralizing antibodies PGT121 and 10-1074, both of which are also dependent on the presence of the gp120 N332 glycan.     

Investigation of Human Cyclooxygenase-2 Glycosylation Heterogeneity and Protein Expression in Insect and Mammalian Cell Expression Systems

Human cyclooxygenase-2 (hCox-2) is a key enzyme in the biosynthesis of prostaglandins and the target of nonsteroidal anti-inflammatory drugs. Recombinant hCox-2 overexpressed in a vaccinia virus (VV)-COS-7 system comprises two glycoforms. Removal of the N-glycosylation consensus sequence at Asn⁵⁸⁰(N580Q and S582A mutants) resulted in the expression of protein comprising a single glycoform, consistent with the partial N-glycosylation at this site in the wild-type (WT) enzyme. The specific cyclooxygenase activities of the purified WT and N580Q mutant were equivalent (40 ± 3 μmol O₂/min/mg) and titrations with diclofenac showed no difference in inhibitor sensitivities of WT and both mutants. Results of the expression of WT and N580Q hCox-2 in aDrosophilaS2 cell system were also consistent with the N-glycosylation at this site, but low levels of activity were obtained. High levels of N-glycosylation heterogeneity are observed in hCox-2 expressed using recombinant baculovirus (BV) in Sf9 cells. Expression of a double N-glycosylation site mutant in Sf9 cells, N580Q/N592Q, resulted in a decrease in glycosylation but no clear decrease in heterogeneity, indicating that the high degree of N-glycosylation heterogeneity observed with the BV-Sf9 system is not due to partial glycosylation of both Asn⁵⁸⁰and Asn⁵⁹². N-linked oligosaccharide profiling of purified VV and BV WT and S582A mutant hCox-2 showed the presence of high mannose structures, (Man)_n(GlcNAc)₂,n= 9, 8, 7, 6. The S582A mutant was the most homogeneous with (Man)₉(GlcNAc)₂comprising greater than 50% of oligosaccharides present. Analysis of purified VV WT and S582A mutant hCox-2 by liquid chromatography–electrospray ionization–mass spectrometry showed an envelope of peaks separated by approximately 160 Da, corresponding to differences of a single monosaccharide. The difference between the highest mass peaks of the two envelopes, of approximately 1500 Da, is consistent with the wild-type enzyme containing an additional high mannose oligosaccharide.     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

The production and characterization of monoclonal antibodies against enkephalins

The hybridoma technology of Kohler and Milstein (1975) was utilized to produce monoclonal antibodies against the enkephalins. Two hybridomas, AD4 and DB4, produced monoclonal antibodies of the IgG type 1 class against Leu⁵-enkephalin that were highly specific for Leu⁵- and Met⁵-enkephalin. AD4 exhibited almost equal reactivity with either Leu⁵- or Met⁵-enkephalin, whereas DB4 exhibited only a 20% cross-reactivity with Met⁵-enkephalin. The IC₅₀ of these monoclonal antibodies were approximately two orders of magnitude greater than the IC₅₀ a polyclonal antiserum against enkephalins (A206; Miller et al 1978) used routinely in many immunochemical and immunocytochemical studies.The monoclonal antibodies, AD4 and DB4, exhibited specific sequence and size requirements for binding enkephalin-related peptides. The amino acid sequence Gly-Gly-Phe-Leu or Gly-Gly-Phe-Met was essential for recognition by AD4 and DB4. However, Tyr-Gly-Gly-Phe which lacks Leu or Met in the fifth position did not react with our monoclonal antibodies. Moreover, enkephalin-related peptides in which the enkephalin sequence was situated at the amino terminus and which contained six or more amino acids did not react significantly with AD4 or DB4. In particular, unlike the polyclonal antiserum A206, our monoclonal antibodies do not react with dynorphins 1–6 or 1–13. However, when the monoclonal antibody (AD4) was used to localize immunohistochemically the population of enkephalinergic amacrine cells in the chicken retina, it provided a staining pattern quite comparable to that observed in previous studies (Watt et al., 1983) using the polyclonal enkephalin antiserum A206. This finding therefore demonstrates that the immunoreactive products visualized in the enkephalin-immunoreactive amacrine cells of the chicken retina with the polyclonal antiserum correspond to authentic enkephalin or peptides very closely related to the enkephalins.     

Specificity of anti-peptide antibodies elicited against synthetic peptides mimicking conserved regions of HIV1 envelope glycoprotein

Comparison of HIV1_Bru and HIV2_Rod external envelope glycoprotein sequences enabled us to select ten highly conserved peptide sequences. The corresponding peptides were chemically synthesized, then coupled to bovine serum albumin before injection in rabbits. Although all peptides were immunogenic, only antibodies directed against peptides P1 (amino acid residues 33–55), P22 (418–462), P8 (487–508) and P21 (487–534) were able to interact with significant affinity (K_0.5 about 10⁻⁶ and 10⁻⁸ M) with the native glycoprotein by radioimmunoassay. Noteworthy was the capacity of anti-P1 antibodies to also recognize the glycoprotein of HIV2. Anti-peptide antibodies were tested for their ability to interfere with the gp120-CD4 interaction, membrane fusion and virus replication. Preincubation of gp 120 with antibodies directed to the region previously described as the putative CD4-binding site, P22 (418–462), did not abolish gp120 binding to CD4-positive cells.     

Selective toxicity of neocarzinostatin-monoclonal antibody conjugates to the antigen-bearing human melanoma cell line in vitro

Summary Monoclonal antibodies (IgG₁) against high molecular weight antigen A-1-43 on human melanoma cell line A-375 were successfully linked to the anti-tumour protein neocarzinostatin (NCS) using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). The conjugate retained both the reactivity of the antibody and the toxicity of the drug. The antigen-bearing cell line A-375, antigen-lacking cell line MeWo and normal skin fibroblasts were exposed to NCS-monoclonal antibody conjugates. As negative control, cells were also treated with free NCS and NCS coupled to normal mouse IgG₁ antibodies. Inhibition of ³H-thymidine uptake after treatment was used to measure the biological activity of the cytotoxic drug complex or substance, respectively.Comparing the inhibition dose for 50% uptake (ID₅₀) it was found that the monoclonal antibody-drug complex is about 100 times more toxic for the antigen-bearing cell line than free NCS or normal mouse IgG₁-NCS. This high toxicity is due to a local increase of drug concentration on these cells. With the two cell lines lacking the appropriate antigen no significant differences in the ID₅₀ values were observed. A selectivity factor of 40–50 was obtained by comparing the cytotoxic effect of the monoclonal antibody-NCS conjugate upon the antigen-bearing as opposed to the antigen-lacking cell type. These data demonstrate, that the toxicity of NCS can be directed by monoclonal antibodies to human tumour cells carrying the corresponding surface antigen.     

Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy

NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in ¹⁵N, ¹³C, or ²H. The bacterial expression systems currently in use to obtain isotopic enrichment, however, cannot produce a number of eukaryotic proteins, especially those that require post-translational modifications such as N-linked glycosylation for proper folding or activity. Here, we report the use of an adenovirus vector-based mammalian expression system to produce isotopically enriched ¹⁵N or ¹⁵N/¹³C samples of an outer domain variant of the HIV-1 gp120 envelope glycoprotein with 15 sites of N-linked glycosylation. Yields for the ¹⁵N- and ¹⁵N/¹³C-labeled gp120s after affinity chromatography were 45 and 44 mg/l, respectively, with an average of over 80% isotope incorporation. Recognition of the labeled gp120 by cognate antibodies that recognize complex epitopes showed affinities comparable to the unlabeled protein. NMR spectra, including ¹H-¹⁵N and ¹H-¹³C HSQCs, ¹⁵N-edited NOESY-HSQC, and 3D HNCO, were of high quality, with signal-to-noise consistent with an efficient level of isotope incorporation, and with chemical shift dispersion indicative of a well-folded protein. The exceptional protein yields, good isotope incorporation, and ability to obtain well-folded post-translationally modified proteins make this mammalian system attractive for the production of isotopically enriched eukaryotic proteins for NMR spectroscopy.     

Synthesis of chondroitin sulfate CC and DD tetrasaccharides and interactions with 2H6 and LY111

We synthesized the biotinylated chondroitin sulfate tetrasaccharides CS-CC [-3)βGalNAc6S(1–4)βGlcA(1-]₂ and CS-DD [-3)βGalNAc6S(1–4)βGlcA2S(1-]₂ which possess sulfate groups at O-6 of GalNAc and an additional sulfate group at O-2 of GlcA, respectively. We also analyzed interactions among CS-CC and CS-DD and the antibodies 2H6 and LY111, both of which are known to bind with CS-A, while CS-DD was shown for the first time to bind with both antibodies.     

Characterization of monoclonal antibodies to protoplast membranes of Nicotiana tabacum identified by an enzyme-linked immunosorbent assay

Murine monoclonal antibodies to protoplast membrne antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofluorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodatesensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of M_r 14 kilodaltons (kDa) and a smear of bands of M_r 45–120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equilibrating at a density of approx. 1.12 kg·l^-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman et al. 1986, Planta 167, 452–459) available for the study of plant plasma-membrane structure and function.Abbreviations ELISA Enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Antibody proteases: induction of catalytic response

Most of the data accumulated throughout the years on investigation of catalytic antibodies indicate that their production increases on the background of autoimmune abnormalities. The different approaches to induction of catalytic response toward recombinant gp120 HIV-1 surface protein in mice with various autoimmune pathologies are described. The peptidylphosphonate conjugate containing structural part of gp120 molecule is used for reactive immunization of NZB/NZW F₁, MRL, and SJL mice. The specific modification of heavy and light chains of mouse autoantibodies with Val-Ala-Glu-Glu-Glu-Val-PO(OPh)₂ reactive peptide was demonstrated. Increased proteolytic activity of polyclonal antibodies in SJL mice encouraged us to investigate the production of antigen-specific catalytic antibodies on the background of induced experimental autoimmune encephalomyelitis (EAE). The immunization of autoimmune-prone mice with the engineered fusions containing the fragments of gp120 and encephalitogenic epitope of myelin basic protein (MBP_89-104) was made. The proteolytic activity of polyclonal antibodies isolated from the sera of autoimmune mice immunized by the described antigen was shown. Specific immune response of SJL mice to these antigens was characterized. Polyclonal antibodies purified from sera of the immunized animals revealed proteolytic activity. The antiidiotypic approach to raise the specific proteolytic antibody as an internal image of protease is described. The second order monoclonal antibodies toward subtilisin Carlsberg revealed pronounced proteolytic activity.     

Three major glycoprotein genes of varicella-zoster virus whose products have neutralization epitopes

Varicella-zoster virus (VZV) codes for approximately eight glycosylated polypeptides in infected cell cultures and in virions. To determine the number of serologically distinct glycoprotein gene products encoded by VZV, we have developed murine monoclonal antibodies to purified virions. Of 10 monoclonal antibodies which can immunoprecipitate intracellular VZV antigens and virion glycoproteins, 1 (termed gA) reacted with gp105, 1 (termed gB) reacted with gp115 (intracellular only), gp62, and gp57, and 8 (termed gC) reacted with gp92, gp83, gp52, and gp45. The anti-gA monoclonal antibody neutralized VZV infectivity in the absence of complement. All eight anti-gC monoclonal antibodies neutralized only in the presence of complement. An anti-gB monoclonal antibody obtained from another laboratory also neutralizes in the absence of complement. Since the above reactivities account for all major detectable VZV glycoprotein species, the data strongly suggest that VZV has three major glycoprotein genes which encode glycosylated polypeptides with neutralization epitopes.     

Enhanced antibody-mediated neutralization of HIV-1 variants that are resistant to fusion inhibitors

Background

HIV-1 typically develops resistance to any single antiretroviral agent. Combined anti-retroviral therapy to reduce drug-resistance development is necessary to control HIV-1 infection. Here, to assess the utility of a combination of antibody and fusion inhibitor treatments, we investigated the potency of monoclonal antibodies at neutralizing HIV-1 variants that are resistant to fusion inhibitors.

Results

Mutations that confer resistance to four fusion inhibitors, enfuvirtide, C34, SC34, and SC34EK, were introduced into the envelope of HIV-1_JR-FL, a CCR5-tropic tier 2 strain. Pseudoviruses with these mutations were prepared and used for the assessment of neutralization sensitivity to an array of antibodies. The resulting neutralization data indicate that the potencies of some antibodies, especially of those against the CD4 binding site, V3 loop, and membrane-proximal external region epitopes, were increased by the mutations in gp41 that conferred resistance to the fusion inhibitors. C34-, SC34-, and SC34EK-resistant mutants showed more sensitivity to monoclonal antibodies than enfuvirtide-resistant mutants. An analysis of C34-resistant mutations revealed that the I37K mutation in gp41 HR1 is a key mutation for C34 resistance, low infectivity, neutralization sensitivity, epitope exposure, and slow fusion kinetics. The N126K mutation in the gp41 HR2 domain contributed to C34 resistance and neutralization sensitivity to anti-CD4 binding site antibodies. In the absence of L204I, the effect of N126K was antagonistic to that of I37K. The results of a molecular dynamic simulation of the envelope trimer confirmation suggest that an I37K mutation induces the augmentation of structural fluctuations prominently in the interface between gp41 and gp120. Our observations indicate that the “conformational unmasking” of envelope glycoprotein by an I37K mutation is one of the mechanisms of neutralization sensitivity enhancement. Furthermore, the enhanced neutralization of C34-resistant mutants in vivo was shown by its high rate of neutralization by IgG from HIV patient samples.

Conclusions

Mutations in gp41 that confer fusion inhibitor resistance exert enhanced sensitivity to broad neutralizing antibodies (e.g., VRC01 and 10E8) and other conventional antibodies developed in HIV-1 infected patients. Therefore, next-generation fusion inhibitors and monoclonal antibodies could be a potential combination for future regimens of combined antiretroviral therapy.

     

Purification,characterization and regulation of the synthesis of an Aspergillus nidulans acidic xylanase

An acidic xylanase from a culture filtrate of Aspergillus nidulans grown on oat-spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular mass of 34,000 Da and had an isoelectric point of approximately 3.4. The enzyme was a non-debranching endoxylanase highly specific for xylans. The xylanase showed an optimal activity at pH 6.0 and 56° C and had a Michaelis constant K_m of 0.97 mg oat-spelt xylan (soluble fraction) ml and a maximed reaction velocity (Vmax) of 1,091 mol min^–1 (mg^–1protein)^–1. Using polyclonal antibodies raised against the purified enzyme, the regulation of its synthesis has been studied. The xylanase production is repressed by glucose and induced by oat-spelt xylan, arabinoxylan, 4-O-methylglucurono-xylan, birchwood xylan and xylose.