71 research outputs found
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
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
Coupling of Site-Specific DNA Binding to Protein Dimerization in Assembly of the Biotin Repressor−Biotin Operator Complex †
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