Biomedical subjects

Antonio Nakouzi

Publications and source records attributed to Antonio Nakouzi.

8 recordsLinked to original sources

PubMed2006

Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport.

The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of C. neoformans whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the capsule network to reach the extracellular environment.

Animals↗

PubMed2006

A monoclonal antibody to Bacillus anthracis protective antigen defines a neutralizing epitope in domain 1.

Antibody (Ab) responses to Bacillus anthracis toxins are protective, but relatively few protective monoclonal antibodies (MAbs) have been reported. Protective antigen (PA) is essential for the action of B. anthracis lethal toxin (LeTx) and edema toxin. In this study, we generated two MAbs to PA, MAbs 7.5G and 10F4. These MAbs did not compete for binding to PA, consistent with specificities for different epitopes. The MAbs were tested for their ability to protect a monolayer of cultured macrophages against toxin-mediated cytotoxicity. MAb 7.5G, the most-neutralizing MAb, bound to domain 1 of PA and reduced LeTx toxicity in BALB/c mice. Remarkably, MAb 7.5G provided protection without blocking the binding of PA or lethal factor or the formation of the PA heptamer complex. However, MAb 7.5G slowed the proteolytic digestion of PA by furin in vitro, suggesting a potential mechanism for Ab-mediated protection. These observations indicate that some Abs to domain 1 can contribute to host protection.

Animals↗

PubMed2005

Structural and functional characterization of glycosylation in an immunoglobulin G1 to Cryptococcus neoformans glucuronoxylomannan.

Analysis of the N-linked oligosaccharides of the murine IgG1 monoclonal antibody (mAb) to Cryptococcus neoformans by LC/MS revealed five different core fucosylated, biantennary complex-type oligosaccharides at Asn-293, with the major species being a mono-galactosylated oligosaccharide with the glycosyl composition of Hex4HexNAc4Fuc (39% of the total glycan pool). The primary sequence predicted from nucleic acid sequencing differed from that measured by mass spectrometry at position 33 (ASN to ASP), a finding that may represent post-translational modification caused by spontaneous ASP deamination. Analysis of mAb 18B7 from three hybridoma clones revealed the same heterogenous N-glycan pattern, indicating that diversity in oligosaccharide structures originated from individual cells. The binding of native and de-glycosylated mAb 18B7 to cryptococcal Ag was comparable but the de-glycosylated 18B7 had shorter serum half-life and did not activate complement (C). De-glycosylated mAb 18B7 was opsonic for C. neoformans with murine macrophages through a mechanism that involved C-independent ingestion through the C receptor. Passive administration of de-glycosylated mAb 18B7 mediated comparable protective efficacy to the native mAb in mice with lethal infection. The results imply that the contribution of N-glycan structure to immunoglobulin function varies depending on the Ag-Ab system.

Amino Acid Sequence↗

PubMed2005

Synthesis and immunological studies of glycoconjugates of Cryptococcus neoformans capsular glucuronoxylomannan oligosaccharide structures.

Antibodies to the glucuronoxylomannan (GXM) component of the polysaccharide capsule of Cryptococcus neoformans are protective and GXM-protein conjugate vaccines can elicit protective immune responses. We report the synthesis of a heptasaccharide oligosaccharide representing the putative dominant motif of serotype A GXM and demonstrate that it is recognized by some monoclonal antibodies (mAbs) generated to GXM. Conjugation of the heptasaccharide to human serum albumin (HSA) resulted in an immunogenic compound that elicited high-titer IgG responses in mice when given with complete Freund's adjuvant. The antibody response elicited by the oligosaccharide conjugate vaccine had characteristics of a T-cell-dependent response. The availability of an immunogenic oligosaccharide representing a structural motif of GXM will prove useful in studies of antibody epitope specificity and represents a potential synthetic oligosaccharide vaccine against this fungal pathogen.

Animals↗

PubMed2003

Ionizing radiation delivered by specific antibody is therapeutic against a fungal infection.

There is an urgent need for new antimicrobial therapies to combat drug resistance, new pathogens, and the relative inefficacy of current therapy in compromised hosts. Ionizing radiation can kill microorganisms quickly and efficiently, but this modality has not been exploited as a therapeutic antimicrobial strategy. We have developed methods to target ionizing radiation to a fungal cell by labeling a specific mAb with the therapeutic radioisotopes Rhenium-188 and Bismuth-213. Radiolabeled antibody killed cells of human pathogenic fungus Cryptococcus neoformans in vitro, thus converting an antibody with no inherent antifungal activity into a microbicidal molecule. Administration of radiolabeled antibody to mice with C. neoformans infection delivered 213Bi and 188Re to the sites of infection, reduced their organ fungal burden, and significantly prolonged their survival without apparent toxicity. This study establishes the principle that targeted radiation can be used for the therapy of an infectious disease, and suggests that it may have wide applicability as an antimicrobial strategy.

Animals↗

PubMed2003

Site-specific characterization of the N-linked oligosaccharides of a murine immunoglobulin M by high-performance liquid chromatography/electrospray mass spectrometry.

Immunoglobulin M is an especially important product of the immune system because it plays a critical role in early protection against infections. In this report, the glycosylation pattern of the protective murine monoclonal IgM 12A1 to Cryptococcus neoformans polysaccharide was analyzed by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Peptide mapping studies covering 88% of the deduced amino acid sequence indicated that of the six potential N-glycosylation sites in this antibody only five were utilized, as the tryptic peptide derived from monoclonal IgM 12A1 containing Asn-260 was recovered without carbohydrates. The oligosaccharide side chains of monoclonal IgM 12A1 were characterized at each of the N-glycosylation sites. Asn-166 possessed 20 monosialylated and nonsialylated, and fucosylated and nonfucosylated complex- and hybrid-type oligosaccharides and one high-mannose-type oligosaccharide. Thirteen oligosaccharides were attached to the site at Asn-401, including six complex-type, four hybrid-type, and three high-mannose-type oligosaccharides. Twelve hybrid-type oligosaccharides were attached to Asn-378, three of which had terminal sialic acids. Eleven hybrid-type oligosaccharides were attached to Asn-331, seven of which had terminal sialic acids. Only two high-mannose type oligosaccharides were attached to Asn-363. These results indicated great complexity in the structure and composition of oligosaccharides attached to individual IgM glycosylation sites.

Amino Acid Sequence↗

PubMed2003

The function of conserved amino acids in or near the complementarity determining regions for related antibodies to Cryptococcus neoformans glucuronoxylomannan.

Most monoclonal antibodies (mAbs) to Cryptococcus neoformans glucuronoxylomannan (GXM) antigen (Ag) are grouped as Class II based on usage of V(H)7183, Vkappa5.1, J(H)2, and Jkappa1 gene elements. Comparative analysis of 43 Class II mAbs revealed conservation of I51, G54, and D61 in heavy-chain variable region (V(H)) complementarity determining region 2 (CDR2), and R95 and D96 in CDR3. Furthermore, position 100b (Kabat numbering scheme) in CDR3 always had an aromatic amino acid (aa) and F was found at position 100c in 96% of mAbs. The function of these conserved residues for binding to GXM and peptide mimetics, and idiotype (Id) structure, was investigated using site-directed mutagenesis. In addition, we mutated W36 and V37 in the second framework. Mutations W36A, Y100bA, and F100cA interfered with antibody (Ab) secretion, but not assembly, and cytoplasmic Ab bound to GXM and Id mAbs. In contrast, mutations V37A, I51A, G54A, and D61A did not affect assembly, secretion, or binding to GXM. Mutating the R95-D96 motif in CDR3 to DR, DD, RR, RA, AD, KD, HD, RE, RN or AA revealed that the positive charge at position 95 was essential for binding GXM, whereas the negative charge at position 96 could be substituted for a non-charged aa. Our results: (1) extend the concept that CDR3 diversity is essential for Ag and Id specificity to a polysaccharide-binding Ab; (2) show that aa conservation in CDRs does not imply a requirement for Ag binding; (3) establish a role for W36 in secretion; and (4) demonstrate that aa motifs used for binding GXM and peptide mimetics can differ.

Antibodies↗

PubMed2002

Isotype can affect the fine specificity of an antibody for a polysaccharide antigen.

Ab specificity is determined by V region sequence. The murine Mab 18B7 (IgG1) binds to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan and produces annular immunofluorescence (IF) on yeast cells. The heavy and light V regions of 18B7 were expressed with the human C regions micro, gamma 1, gamma 2, gamma 3, gamma 4, and alpha1, and the specificity and binding properties of these mouse-human chimeric (ch) Abs was determined. The chIgG1, chIgG2, chIgG4, and the chIgA produced annular IF, whereas the IgM and IgG3 produced punctate IF, despite identical V region sequences. Competition experiments with murine Abs that competed with mAb 18B7 and binding assays to peptide mimetics of glucuronoxylomannan provided additional evidence for altered specificity in some of the ch Abs. Expression of 18B7 heavy V region with murine micro C region produced IgM with a punctate IF, indicating that a change in fine specificity also accompanied the change from murine IgG1 to IgM. Our results show that Ab fine specificity can be a function of isotype. This phenomenon may be most apparent for Abs that bind to Ag with repeating epitopes, such as polysaccharides, where the quarternary structure of the Ag-Ab complex may be influenced by such constraints as Fab-Fab angles, Fc-Fc interactions, Ab size, and solvent accessibility to exposed surfaces. Alterations in Ab fine specificity following isotype change could have important implications for current concepts on the generation of secondary Ab responses to certain Ags and for the isotype preference observed in Abs to polysaccharides.

Animals↗