A Folate Receptor Beta-Specific Human Monoclonal Antibody Recognizes Activated Macrophage of Rheumatoid Patients and Mediates Antibody-Dependent Cell-Mediated Cytotoxicity.

Introduction Folate receptor beta (FRβ) is only detectable in placenta and limited to some hematopoietic cells of myeloid lineage in healthy people. Studies have indicated that FRβ is over-expressed in activated macrophages in autoimmune diseases and some cancer cells. In this study we aimed to develop an FRβ-specific human monoclonal antibody (mAb) that could be used as a therapeutic agent to treat rheumatoid arthritis and other autoimmune diseases, as well as FRβ positive cancers. Methods Functional recombinant FRβ protein was produced in insect cells and used as antigen to isolate a mAb, m909, from a human naïve Fab phage display library. Binding of Fab and IgG1 m909 to FRβ was measured by ELISA, surface plasmon resonance, immune fluorescence staining, and flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated with FRβ positive CHO cells as target cells and isolated peripheral blood monocytes as effector cells in an in vitroassay. Results Fab m909 bound with relatively high affinity (equilibrium dissociation constant 57 nM) to FRβ. The IgG1 m909 showed much higher (femtomolar) avidity as measured by ELISA, and it bound to FRβ positive cells in a dose-dependent manner, but not to parental FRβ negative cells. m909 did not compete with folate for the binding to FRβ on cells. m909 was not only able to select FRβ positive, activated macrophages from synovial fluid cells of arthritis patients as efficiently as folate, but also able to mediate ADCC in FRβ positive cells. Conclusions Unlike folate-drug conjugates, m909 selectively binds to FRβ, does not recognize FRα, and has at least one effector function. m909 alone has potential to eliminate FRβ positive cells. Because m909 does not compete with folate for receptor binding, it can be used with folate-drug conjugates in a combination therapy. m909 can also be a valuable research reagent

454 antibody sequencing - error characterization and correction

BACKGROUND: 454 sequencing is currently the method of choice for sequencing of antibody repertoires and libraries containing large numbers (10(6 )to 10(12)) of different molecules with similar frameworks and variable regions which poses significant challenges for identifying sequencing errors. Identification and correction of sequencing errors in such mixtures is especially important for the exploration of complex maturation pathways and identification of putative germline predecessors of highly somatically mutated antibodies. To quantify and correct errors incorporated in 454 antibody sequencing, we sequenced six antibodies at different known concentrations twice over and compared them with the corresponding known sequences as determined by standard Sanger sequencing. RESULTS: We found that 454 antibody sequencing could lead to approximately 20% incorrect reads due to insertions that were mostly found at shorter homopolymer regions of 2-3 nucleotide length, and less so by insertions, deletions and other variants at random sites. Correction of errors might reduce this population of erroneous reads down to 5-10%. However, there are a certain number of errors accounting for 4-8% of the total reads that could not be corrected unless several repeated sequencing is performed, although this may not be possible for large diverse libraries and repertoires including complete sets of antibodies (antibodyomes). CONCLUSIONS: The experimental test procedure carried out for assessing 454 antibody sequencing errors reveals high (up to 20%) incorrect reads; the errors can be reduced down to 5-10% but not less which suggests the use of caution to avoid false discovery of antibody variants and diversity

Ligand Specificity of Group I Biotin Protein Ligase of Mycobacterium tuberculosis

BACKGROUND: Fatty acids are indispensable constituents of mycolic acids that impart toughness & permeability barrier to the cell envelope of M. tuberculosis. Biotin is an essential co-factor for acetyl-CoA carboxylase (ACC) the enzyme involved in the synthesis of malonyl-CoA, a committed precursor, needed for fatty acid synthesis. Biotin carboxyl carrier protein (BCCP) provides the co-factor for catalytic activity of ACC. METHODOLOGY/PRINCIPAL FINDINGS: BPL/BirA (Biotin Protein Ligase), and its substrate, biotin carboxyl carrier protein (BCCP) of Mycobacterium tuberculosis (Mt) were cloned and expressed in E. coli BL21. In contrast to EcBirA and PhBPL, the approximately 29.5 kDa MtBPL exists as a monomer in native, biotin and bio-5'AMP liganded forms. This was confirmed by molecular weight profiling by gel filtration on Superdex S-200 and Dynamic Light Scattering (DLS). Computational docking of biotin and bio-5'AMP to MtBPL show that adenylation alters the contact residues for biotin. MtBPL forms 11 H-bonds with biotin, relative to 35 with bio-5'AMP. Docking simulations also suggest that bio-5'AMP hydrogen bonds to the conserved 'GRGRRG' sequence but not biotin. The enzyme catalyzed transfer of biotin to BCCP was confirmed by incorporation of radioactive biotin and by Avidin blot. The K(m) for BCCP was approximately 5.2 microM and approximately 420 nM for biotin. MtBPL has low affinity (K(b) = 1.06x10(-6) M) for biotin relative to EcBirA but their K(m) are almost comparable suggesting that while the major function of MtBPL is biotinylation of BCCP, tight binding of biotin/bio-5'AMP by EcBirA is channeled for its repressor activity. CONCLUSIONS/SIGNIFICANCE: These studies thus open up avenues for understanding the unique features of MtBPL and the role it plays in biotin utilization in M. tuberculosis

Diversity in Functional Organization of Class I and Class II Biotin Protein Ligase

The cell envelope of Mycobacterium tuberculosis (M.tuberculosis) is composed of a variety of lipids including mycolic acids, sulpholipids, lipoarabinomannans, etc., which impart rigidity crucial for its survival and pathogenesis. Acyl CoA carboxylase (ACC) provides malonyl-CoA and methylmalonyl-CoA, committed precursors for fatty acid and essential for mycolic acid synthesis respectively. Biotin Protein Ligase (BPL/BirA) activates apo-biotin carboxyl carrier protein (BCCP) by biotinylating it to an active holo-BCCP. A minimal peptide (Schatz), an efficient substrate for Escherichia coli BirA, failed to serve as substrate for M. tuberculosis Biotin Protein Ligase (MtBPL). MtBPL specifically biotinylates homologous BCCP domain, MtBCCP87, but not EcBCCP87. This is a unique feature of MtBPL as EcBirA lacks such a stringent substrate specificity. This feature is also reflected in the lack of self/promiscuous biotinylation by MtBPL. The N-terminus/HTH domain of EcBirA has the self-biotinable lysine residue that is inhibited in the presence of Schatz peptide, a peptide designed to act as a universal acceptor for EcBirA. This suggests that when biotin is limiting, EcBirA preferentially catalyzes, biotinylation of BCCP over self-biotinylation. R118G mutant of EcBirA showed enhanced self and promiscuous biotinylation but its homologue, R69A MtBPL did not exhibit these properties. The catalytic domain of MtBPL was characterized further by limited proteolysis. Holo-MtBPL is protected from proteolysis by biotinyl-5′ AMP, an intermediate of MtBPL catalyzed reaction. In contrast, apo-MtBPL is completely digested by trypsin within 20 min of co-incubation. Substrate selectivity and inability to promote self biotinylation are exquisite features of MtBPL and are a consequence of the unique molecular mechanism of an enzyme adapted for the high turnover of fatty acid biosynthesis

Nonenzymatic biotinylation of a biotin carboxyl carrier protein: Unusual reactivity of the physiological target lysine

Enzyme-catalyzed addition of biotin to proteins is highly specific. In any single organism one or a small number of proteins are biotinylated and only a single lysine on each of these proteins is modified. A detailed understanding of the structural basis for the selective biotinylation process has not yet been elucidated. Recently certain mutants of the Escherichia coli biotin protein ligase have been shown to mediate “promiscuous” biotinylation of proteins. It was suggested that the reaction involved diffusion of a reactive activated biotin intermediate, biotinoyl-5′-AMP, with nonspecific proteins. In this work the reactivity of this chemically synthesized intermediate toward the natural target of enzymatic biotinylation, the biotin carboxyl carrier protein, was investigated. The results indicate that the intermediate does, indeed, react with target protein, albeit at a significantly slower rate than the enzyme-catalyzed process. Surprisingly, analysis of the products of nonenzymatic biotinylation indicates that of five lysine residues in the protein only the physiological target side chain is modified. These results indicate that either the environment of this lysine residue or its intrinsic properties render it highly reactive to nonenzymatic biotinylation mediated by biotinoyl-5′-AMP. This reactivity may be important for its selective biotinylation in vivo