Nobuko Hamaguchi

Aptamer-based biosensor arrays for detection and quantification of biological macromolecules

We have developed a chip-based biosensor for multiplex analysis of protein analytes. The biosensor utilizes immobilized DNA and RNA aptamers, selected against several different protein targets, to simultaneously detect and quantify levels of individual proteins in complex biological mixtures. Aptamers were each fluorescently labeled and immobilized on a glass substrate. Fluorescence polarization anisotropy was used for solid- and solution-phase measurements of target protein binding. We show that solid-phase aptamer-protein interactions recapitulate binding interactions seen in solution. Furthermore, we demonstrate specific detection and quantitation of cancer-associated proteins (inosine monophosphate dehydrogenase II, vascular endothelial factor, basic fibroblast growth factor) in the context of human serum and in cellular extracts. It is expected that this technology could speed diagnosis of cancer by enabling direct detection of the expression and modification of proteins closely correlated with disease.

Changing Residue 338 in Human Factor IX from Arginine to Alanine Causes an Increase in Catalytic Activity

This study was designed to identify functionally important factor IX (FIX) residues. Using recombinant techniques and cell culture, we produced a mutant FIX with arginine at 338 changed to alanine (R338A-FIX). This molecule had approximately 3 times greater clotting activity than that of wild type FIX (wt-FIX) in the activated partial thromboplastin assay. R338A-FIX reacted normally with a panel of three FIX specific monoclonal antibodies and migrated on sodium dodecyl sulfate-polyacrylamide gels indistinguishably from wt-FIX. Using functional assays, we determined that R338A-FIXa’sK d for factor VIIIa (FVIIIa) was similar to that of wt-FIXa. Our kinetic analysis, using factor X as substrate, indicated that the mutation’s major effects were a 3-fold increase ink cat and a 2-fold decrease inK m both manifested only in the presence of FVIIIa. R338A-FIXa’s increased catalytic efficiency did not result from ablation of a thrombin sensitive site, reported to occur at arginine 338, since in our assays the thrombin inhibitor, hirudin, had no effect on activity of either wt-FIXa or R338A-FIXa. R338A-FIXa and wt-FIXa had equal activity, with or without FVIIIa, toward the synthetic substrate, methylsulfonyl-d-cyclohexylglycyl-arginine-p-nitroanilide. Interestingly, R338A-FIXa had reduced affinity for heparin. Therefore, we propose that R338A-FIXa’s increased activity is not due to an allosteric effect on the active site, but that the Arg-338 residue is part of an exosite that binds both factor X and the mucopolysaccharide, heparin.

IMP Dehydrogenase Type 1 Associates with Polyribosomes Translating Rhodopsin mRNA

IMP dehydrogenase (IMPDH) catalyzes the pivotal step in guanine nucleotide biosynthesis. Here we show that both IMPDH type 1 (IMPDH1) and IMPDH type 2 are associated with polyribosomes, suggesting that these housekeeping proteins have an unanticipated role in translation regulation. This interaction is mediated by the subdomain, a region of disputed function that is the site of mutations that cause retinal degeneration. The retinal isoforms of IMPDH1 also associate with polyribosomes. The most common disease-causing mutation, D226N, disrupts the polyribosome association of at least one retinal IMPDH1 isoform. Finally, we find that IMPDH1 is associated with polyribosomes containing rhodopsin mRNA. Because any perturbation of rhodopsin expression can trigger apoptosis in photoreceptor cells, these observations suggest a likely pathological mechanism for IMPDH1-mediated hereditary blindness. We propose that IMPDH coordinates the translation of a set of mRNAs, perhaps by modulating localization or degradation.

Generation of a novel factor IX with augmented clotting activities in vitro and in vivo

Summary.  Background: Hemophilia B is an X‐linked inherited disorder caused by the lack of functional factor IX (FIX). Currently, treatment of hemophilia B is performed by intravenous infusion of plasma‐derived or recombinant FIX. Objective: In an effort to reduce factor usage and cost, we investigated the potential use of FIX variants with enhanced specific clotting activity. Methods: Seven recombinant FIX variants using alanine replacement were generated and assayed for their activity in vitro and in vivo. Results: One variant containing three substitutions (V86A/E277A/R338A, FIX‐Triple) exhibited 13‐fold higher specific clotting activity and a 10‐fold increased affinity for human FVIIIa compared with FIX‐wild‐type (FIX‐WT) and was thus investigated systematically in vivo. Liver‐specific FIX‐Triple gene expression following hydrodynamic plasmid delivery revealed a 3.5‐fold higher specific clotting activity compared with FIX‐WT. Human FIX‐Triple and FIX‐WT knock‐in mice were generated and it was confirmed that FIX‐Triple has 7‐fold higher specific clotting activity than FIX‐WT under normal physiological conditions. Protein infusion of FIX‐Triple into hemophilia B mice resulted in greater improvement of hemostasis than that achieved with FIX‐WT. Moreover, tail‐vein administration of a serotype 8 recombinant Adeno‐associated vector (AAV8) expressing either FIX‐WT or FIX‐Triple in hemophilia B mice demonstrated a 7‐fold higher specific clotting activity of FIX‐Triple than FIX‐WT. Conclusions: Our results indicate that the FIX‐Triple variant exhibits significantly enhanced clotting activity relative to FIX‐WT due to tighter binding to FVIIIa, as demonstrated both in vitro and in vivo. Therefore, FIX‐Triple is a good candidate for further evaluation in protein replacement therapy as well as gene‐based therapeutic strategies.

Characterisation of factor IX with a glycine-to-valine missense mutation at residue 190 in a patient with severe haemophilia B

A patient with severe haemophilia B with a glycine-to-valine missense mutation at residue 190 (c25, chymotrypsin numbering) in factor IX (FIX; FIX-G190V or FIX-FuChou) had <1% of normal FIX clotting activity and 36% of normal FIX antigen levels (cross-reacting material- reduced, CRMr). Residue 190 in the C-terminal protease domain of human FIX is highly conserved in mammalian species and the serine protease family, suggesting that it has an indispensable role in protein function. To explore the pathological mechanism by which this mutation contributes to dysfunction of the FIX molecule, we functionally characterised FIX-G190V in vitro and in vivo. Liver-specific FIX-G190V gene expression following hydrodynamic plasmid delivery into haemophilia B mice revealed a 5.7-fold reduction in specific clotting activity compared with FIX-WT (wild type) and a two-fold decrease in plasma FIX-G190V concentration. Pulse-chase analysis demonstrated that FIX-G190V was secreted at a significantly slower rate than was FIX-WT. Purified FIX-G190V and FIX-WT displayed normal calcium-dependent conformational changes as shown by intrinsic fluorescence quenching. The in vivo half-lives of FIX-G190V and FIX-WT were indistinguishable. FIX-G190V was, however, more readily degraded than FIX-WT, especially after being activated by the active form of FXI. The vulnerable sites were mapped to the peptide bonds at Arg¹¹⁶-Leu¹¹⁷, Lys²⁶⁵-Tyr²⁶⁶, Arg³²⁷-Val³²⁸, and Arg³³⁸-Ser³³⁹, which are in the exposed loops of the FIX molecule. Also, failure of FXIa-activated FIX-G190V to bind p-aminobenzamidine indicated an abnormal conformation of the active-site pocket. Thus, the mutation at residue 190 of FIX may result in protein misfolding that affects secretion, clotting function, and hydrolysis.