The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6.

The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure-function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis.

Immune focusing and enhanced neutralization induced by HIV-1 gp140 chemical cross-linking.

Experimental vaccine antigens based upon the HIV-1 envelope glycoproteins (Env) have failed to induce neutralizing antibodies (NAbs) against the majority of circulating viral strains as a result of antibody evasion mechanisms, including amino acid variability and conformational instability. A potential vaccine design strategy is to stabilize Env, thereby focusing antibody responses on constitutively exposed, conserved surfaces, such as the CD4 binding site (CD4bs). Here, we show that a largely trimeric form of soluble Env can be stably cross-linked with glutaraldehyde (GLA) without global modification of antigenicity. Cross-linking largely conserved binding of all potent broadly neutralizing antibodies (bNAbs) tested, including CD4bs-specific VRC01 and HJ16, but reduced binding of several non- or weakly neutralizing antibodies and soluble CD4 (sCD4). Adjuvanted administration of cross-linked or unmodified gp140 to rabbits generated indistinguishable total gp140-specific serum IgG binding titers. However, sera from animals receiving cross-linked gp140 showed significantly increased CD4bs-specific antibody binding compared to animals receiving unmodified gp140. Moreover, peptide mapping of sera from animals receiving cross-linked gp140 revealed increased binding to gp120 C1 and V1V2 regions. Finally, neutralization titers were significantly elevated in sera from animals receiving cross-linked gp140 rather than unmodified gp140. We conclude that cross-linking favors antigen stability, imparts antigenic modifications that selectively refocus antibody specificity and improves induction of NAbs, and might be a useful strategy for future vaccine design.

Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin.

Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides mimicking defined epitopes of the bone modulator protein sclerostin, which has been identified as a negative regulator of the Wnt pathway. For a fast exploration of activity defining epitopes, we produced a set of synthetic peptide constructs mimicking native sclerostin, in which intervening loops from the cystine-knot protein sclerostin were truncated and whose sequences were optimized for fast and productive refolding. We found that the second loop within the cystine knot could be replaced by unnatural sequences, both speeding up folding, and increasing yield. Subsequently, we used these constructs to pan the HuCAL phage display library for antibodies capable of binding the native protein, thereby restricting recognition to the desired epitope regions. It is shown that the antibodies that were obtained recognize a complex epitope in the protein that cannot be mimicked with linear peptides. Antibodies selected against peptides show similar recognition specificity and potency as compared with antibodies obtained from full-length recombinant protein.

Molecular and Biochemical Characterization of Rat epsilon -N-Trimethyllysine Hydroxylase, the First Enzyme of Carnitine Biosynthesis.

epsilon-N-Trimethyllysine hydroxylase (EC ) is the first enzyme in the biosynthetic pathway of l-carnitine and catalyzes the formation of beta-hydroxy-N-epsilon-trimethyllysine from epsilon-N-trimethyllysine, a reaction dependent on alpha-ketoglutarate, Fe(2+), and oxygen. We purified the enzyme from rat kidney and sequenced two internal peptides by quadrupole-time-of-flight mass spectroscopy. The peptide sequences were used to search the Expressed Sequence Tag data base, which led to the identification of a rat cDNA of 1218 base pairs encoding a polypeptide of 405 amino acids with a calculated molecular mass of 47.5 kDa. Using the rat sequence we also identified the homologous cDNAs from human and mouse. Heterologous expression of both the rat and human cDNAs in COS cells confirmed that they encode epsilon-N-trimethyllysine hydroxylase. Subcellular fractionation studies revealed that the rat enzyme is localized exclusively in mitochondria. Expression studies in yeast indicated that the rat enzyme is synthesized as a 47.5-kDa precursor and subsequently processed to a mature protein of 43 kDa, presumably upon import in mitochondria. The Michaelis-Menten constants of the purified rat enzyme for trimethyllysine, alpha-ketoglutarate, and Fe(2+) were 1.1 mm, 109 microm, and 54 microm, respectively. Both gel filtration and blue native polyacrylamide gel electrophoresis analysis showed that the native enzyme has a mass of approximately 87 kDa, indicating that in rat epsilon-N-trimethyllysine hydroxylase is a homodimer.

A new crosslinker for mass spectrometric analysis of the quaternary structure of protein complexes.

Mass spectrometric structural analysis of crosslinked peptides is a powerful method to elucidate the spatial arrangement of polypeptides in protein complexes. Our aim is to develop bifunctional crosslinkers that, after crosslinking protein complexes followed by proteolytic digestion, give rise to crosslinked peptides that can be readily tracked down by mass spectrometry. To this end we synthesized the crosslinker N-benzyliminodiacetoyloxysuccinimid (BID), which yields stable benzyl cation marker ions upon low-energy collision-induced dissociation (CID) tandem mass spectrometry. Sensitive detection of the marker ion upon low-energy CID is demonstrated with different BID-crosslinked peptide preparations. With BID it becomes possible to retrieve crosslinked and crosslinker-adducted peptides, without the necessity of purifying crosslinked peptides prior to identification. The basic design of this crosslinker can be varied upon, in order to meet specific crosslinking needs.

Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins.

Prohibitins are ubiquitous, abundant and evolutionarily strongly conserved proteins that play a role in important cellular processes. Using blue native electrophoresis we have demonstrated that human prohibitin and Bap37 together form a large complex in the mitochondrial inner membrane. This complex is similar in size to the yeast complex formed by the homologues Phb1p and Phb2p. In yeast, levels of this complex are increased on co-overexpression of both Phb1p and Phb2p, suggesting that these two proteins are the only components of the complex. Pulse-chase experiments with mitochondria isolated from phb1/phb2-null and PHB1/2 overexpressing cells show that the Phb1/2 complex is able to stabilize newly synthesized mitochondrial translation products. This stabilization probably occurs through a direct interaction because association of mitochondrial translation products with the Phb1/2 complex could be demonstrated. The fact that Phb1/2 is a large multimeric complex, which provides protection of native peptides against proteolysis, suggests a functional homology with protein chaperones with respect to their ability to hold and prevent misfolding of newly synthesized proteins.

In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells.

The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. The D198A amino acid replacement abolished detectable 3'-5' (proofreading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous culture. Further culture resulted in the selection of cells with an inactivated mutator polymerase, and a reduced mutation load in mtDNA. Transient expression of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial DNA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression. These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities.