Silicase, an Enzyme Which Degrades Biogenous Amorphous Silica: Contribution to the Metabolism of Silica Deposition in the Demosponge Suberites domuncula

Carbonic anhydrases (CAs I-VII) are products of a gene family that encodes seven isozymes and several homologous, CA- related proteins. All seven isozymes have been cloned, sequenced, and mapped, and the intron-exon organization of five genes established. They differ in subcellular localizations, being cytoplasmic (CA I, II, III, and VII), GPI-anchored to plasma membranes of specialized epithelial and endothelial cells (CA IV), in mitochondria (CA V), or in salivary secretions (CA VI). They also differ in kinetic properties, susceptibility to inhibitors, and tissue-specific distribution. Structural and kinetic studies of recombinant natural and mutant CAs have greatly increased our understanding of the structural requirements for catalysis. Studies of the effects of CA inhibitors over many years have implicated CAs in a variety of physiological processes. Analyses of human and animal CA deficiencies provide unique opportunities to understand the individual contributions of different isozymes to these processes.

Nanoscale control of the polymerization of silicon and oxygen determines the structures and properties of a wide range of siloxane-based materials, including glasses, ceramics, mesoporous molecular sieves and catalysts, elastomers, resins, insulators, optical coatings, and photoluminescent polymers. In contrast to anthropogenic and geological syntheses of these materials that require extremes of temperature, pressure, or pH, living systems produce a remarkable diversity of nanostructured silicates at ambient temperatures and pressures and at near-neutral pH. We show here that the protein filaments and their constituent subunits comprising the axial cores of silica spicules in a marine sponge chemically and spatially direct the polymerization of silica and silicone polymer networks from the corresponding alkoxide substrates in vitro, under conditions in which such syntheses otherwise require either an acid or base catalyst. Homology of the principal protein to the well known enzyme cathepsin L points to a possible reaction mechanism that is supported by recent site-directed mutagenesis experiments. The catalytic activity of the "silicatein" (silica protein) molecule suggests new routes to the synthesis of silicon-based materials.

The cytoplasmic carboxyl-terminal domain of AE1, the plasma membrane chloride/bicarbonate exchanger of erythrocytes, contains a binding site for carbonic anhydrase II (CAII). To examine the physiological role of the AE1/CAII interaction, anion exchange activity of transfected HEK293 cells was monitored by following the changes in intracellular pH associated with AE1-mediated bicarbonate transport. AE1-mediated chloride/bicarbonate exchange was reduced 50-60% by inhibition of endogenous carbonic anhydrase with acetazolamide, which indicates that CAII activity is required for full anion transport activity. AE1 mutants, unable to bind CAII, had significantly lower transport activity than wild-type AE1 (10% of wild-type activity), suggesting that a direct interaction was required. To determine the effect of displacement of endogenous wild-type CAII from its binding site on AE1, AE1-transfected HEK293 cells were co-transfected with cDNA for a functionally inactive CAII mutant, V143Y. AE1 activity was maximally inhibited 61 +/- 4% in the presence of V143Y CAII. A similar effect of V143Y CAII was found for AE2 and AE3cardiac anion exchanger isoforms. We conclude that the binding of CAII to the AE1 carboxyl-terminus potentiates anion transport activity and allows for maximal transport. The interaction of CAII with AE1 forms a transport metabolon, a membrane protein complex involved in regulation of bicarbonate metabolism and transport.