A novel anti-aldolase C antibody specifically interacts with residues 85–102 of the protein

Aldolase C is a brain-specific glycolytic isozyme whose complete repertoire of functions are obscure. This lack of knowledge can be addressed using molecular tools that discriminate the protein from the homologous, ubiquitous paralog aldolase A. The anti-aldolase C antibodies currently available are polyclonal and not highly specific. We obtained the novel monoclonal antibody 9F against human aldolase C, characterized its isoform specificity and tested its performance. First, we investigated the specificity of 9F for aldolase C. Then, using bioinformatic tools coupled to molecular cloning and chemical synthesis approaches, we produced truncated human aldolase C fragments, and assessed 9F binding to these fragments by western blot and ELISA assays. This strategy revealed that residues 85–102 harbor the epitope-containing region recognized by 9F. The efficiency of 9F was demonstrated also for immunoprecipitation assays. Finally, surface plasmon resonance revealed that the protein has a high affinity toward the epitope-containing peptide. Taken together, our findings show that epitope recognition is sequence-driven and is independent of the three-dimensional structure. In conclusion, given its specific molecular interaction, 9F is a novel and powerful tool to investigate aldolase C’s functions in the brain.     

A novel anti-aldolase C antibody specifically interacts with residues 85–102 of the protein

Aldolase C is a brain-specific glycolytic isozyme whose complete repertoire of functions are obscure. This lack of knowledge can be addressed using molecular tools that discriminate the protein from the homologous, ubiquitous paralog aldolase A. The anti-aldolase C antibodies currently available are polyclonal and not highly specific. We obtained the novel monoclonal antibody 9F against human aldolase C, characterized its isoform specificity and tested its performance. First, we investigated the specificity of 9F for aldolase C. Then, using bioinformatic tools coupled to molecular cloning and chemical synthesis approaches, we produced truncated human aldolase C fragments, and assessed 9F binding to these fragments by western blot and ELISA assays. This strategy revealed that residues 85–102 harbor the epitope-containing region recognized by 9F. The efficiency of 9F was demonstrated also for immunoprecipitation assays. Finally, surface plasmon resonance revealed that the protein has a high affinity toward the epitope-containing peptide. Taken together, our findings show that epitope recognition is sequence-driven and is independent of the three-dimensional structure. In conclusion, given its specific molecular interaction, 9F is a novel and powerful tool to investigate aldolase C’s functions in the brain.     

Neuronal expression of enhanced green fluorescent protein directed by 5' flanking sequences of the rat aldolase C gene in transgenic mice

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a combination of distal and proximal 5' flanking sequences, the A+C+0.8 kilobase (kb) pairs fragments, ensured high brain-specific expression in vivo (Skala et al. 1998). We show here that the expression pattern conferred by these sequences, when placed in front of the chloramphenicol acetyltransferase (CAT) or the enhanced green fluorescent protein (EGFP) reporter genes in transgenic mice, is similar to the distribution of the endogenous mRNA and protein. Double immunostaining for neuronal or glial cell-specific markers and for the EGFP protein indicates that the A+C+0.8 kb genomic sequences from the rat aldolase C gene direct a predominant expression in neuronal cells of adult brain.     

Neuronal expression of enhanced green fluorescent protein directed by 5' flanking sequences of the rat aldolase C gene in transgenic mice.

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a combination of distal and proximal 5' flanking sequences, the A + C + 0.8 kilobase (kb) pairs fragments, ensured high brain-specific expression in vivo (Skala et al. 1998). We show here that the expression pattern conferred by these sequences, when placed in front of the chloramphenicol acetyltransferase (CAT) or the enhanced green fluorescent protein (EGFP) reporter genes in transgenic mice, is similar to the distribution of the endogenous mRNA and protein. Double immunostaining for neuronal or glial cell-specific markers and for the EGFP protein indicates that the A + C + 0.8 kb genomic sequences from the rat aldolase C gene direct a predominant expression in neuronal cells of adult brain.     

The complete amino acid sequence of the human aldolase C isozyme derived from genomic clones

The complete protein sequence of the human aldolase C isozyme has been determined from recombinant genomic clones. A genomic fragment of 6673 base pairs was isolated and the DNA sequence determined. Aldolase protein sequences, being highly conserved, allowed the derivation of the sequence of this isozyme by comparison of open reading frames in the genomic DNA to the protein sequence of other human aldolase enzymes. The protein sequence of the third aldolase isozyme found in vertebrates, aldolase C, completes the primary structural determination for this family of isozymes. Overall, the aldolase C isozyme shared 81% amino acid homology with aldolase A and 70% homology with aldolase B. The comparisons with other aldolase isozymes revealed several aldolase C-specific residues which could be involved in its function in the brain. The data indicated that the gene structure of aldolase C is the same as other aldolase genes in birds and mammals, having nine exons separated by eight introns, all in precisely the same positions, only the intron sizes being different. Eight of these exons contain the protein coding region comprised of 363 amino acids. The entire gene is approximately 4 kilobases.     

Converting the guanine phosphoribosyltransferase from Giardia lamblia to a hypoxanthine-guanine phosphoribosyltransferase

Guanine phosphoribosyltransferase from Giardia lamblia, a key enzyme in the purine salvage pathway, is a potential target for anti-giardiasis chemotherapy. Recent structural determination of GPRTase (Shi, W., Munagala, N. R., Wang, C. C., Li, C. M., Tyler, P. C., Furneaux, R. H., Grubmeyer, C., Schramm, V. L., and Almo, S. C. (2000) Biochemistry 39, 6781-6790) showed distinctive features, which could be responsible for its singular guanine specificity. Through characterizing specifically designed site-specific mutants of GPRTase, we identified essential moieties in the active site for substrate binding. Mutating the unusual Tyr-127 of GPRTase to the highly conserved Ile results in 6-fold lower K(m) for guanine. A L186F mutation in GPRTase increased the affinity toward guanine by 3. 3-fold, whereas the corresponding human HGPRTase mutant L192F showed a 33-fold increase in K(m) for guanine. A double mutant (Y127I/K152R) of GPRTase retained the improved binding of guanine and also enabled the enzyme to utilize hypoxanthine as a substrate with a K(m) of 54 +/- 15.5 microm. A triple mutant (Y127I/K152R/L186F) resulted in further increased binding affinity with both guanine and hypoxanthine with the latter showing a lowered K(m) of 29.8 +/- 4.1 microm. Dissociation constants measured by fluorescence quenching showed 6-fold tighter binding of GMP with the triple mutant compared with wild type. Thus, by increasing the binding affinity of 6-oxopurine, we were able to convert the GPRTase to a HGPRTase.     

Fluorescence microscope study of the binding of added C protein to skeletal muscle myofibrils

The binding of extra C protein to rabbit skeletal muscle myofibrils has been investigated by fluorescence microscopy with fluorescein-labeled C protein or unmodified C protein plus fluorescein-labeled anti-C protein. Added C protein binds strongly to the I bands, which is consistent with its binding to F actin in solution (Moos, C., C. M. Mason, J. M. Besterman, I. M. Feng, and J. H. Dubin. 1978. J. Mol. Biol. 124:571-586). Of particular interest, the binding to the I band is calcium regulated: it requires a free calcium ion concentration comparable to that which activates the myofibrillar ATPase. This increases the likelihood that C protein-actin interaction might be physiologically significant. When I band binding is suppressed, binding in the A band becomes evident. It appears to occur particularly near the M line, and possibly at the edges of the A band as well, suggesting that those parts of the thick filaments that lack C protein in vivo may nevertheless be capable of binding added C protein.