Complementary DNA Cloning and Characterization of Pearlin, a New Class of Matrix Protein in the Nacreous Layer of Oyster Pearls.

Calcified shell layer is composed of two polymorphs of CaCO(3), aragonite or calcite, and an organic matrix. The organic matrix consists of EDTA-soluble and insoluble fractions. These fractions are thought to regulate the formation of the elaborate shell structure. After decalcification of powdered pearl with 0.3 M EDTA, an EDTA-insoluble fraction was extracted with 0.3 M EDTA/8 M urea. This extraction step enabled us to purify a new class of EDTA-insoluble protein, Pearlin, almost homogeneously. Pearlin has a molecular weight of about 15 kDa and contains a sulfated mucopolysaccharide. We cloned the complementary DNA coding for Pearlin and deduced its complete amino acid sequence. Sequence analysis reveals that Pearlin is composed of 129 amino acids with a high proportion of Gly (10.8%), Tyr (10.0%), Cys (8.5%), Asn (7.7%), Asp (7.7%), and Arg (7.7%). Northern blot analysis showed that Pearlin messenger RNA was expressed specifically in mantle epithelium. From the sequencing data, Pearlin was shown to be quite different from the fibrous protein rich in Ala and Gly. The function of this protein in biomineralization is discussed.

Production of a hybrid protein consisting of the N-terminal fragment of urokinase and the C-terminal domain of urinary trypsin inhibitor in Escherichia coli.

We have constructed a hybrid protein (ATFHI) consisting of an N-terminal fragment from urokinase (ATF) and HI-8, which is the C-terminal domain of urinary trypsin inhibitor. The fusion genes for the hybrid proteins were engineered by PCR and cloned into expression plasmids. Under the control of the tac promoter, fusion genes were efficiently expressed in Escherichia coli. The hybrid proteins, produced as inclusion bodies in E. coli, were refolded by a dialysis method and purified by ion-exchange chromatography. ATFHI exhibited bifunctional activity related to antimetastatic effects: the urokinase receptor-binding activity of ATF and the inhibitory activity of HI-8 on plasmin.

A carbonic anhydrase from the nacreous layer in oyster pearls.

It is believed that the polymorphism observed in calcium carbonate crystals, such as aragonite and calcite in mollusk shells, is controlled by organic matrix proteins secreted from the mantle epithelia. However, the fine structures of these proteins are still unknown, and to understand the molecular mechanisms of mineralization process, detailed structural analyses of the organic matrix proteins are essential. For this, we have carried out purification, characterization, and cDNA cloning of nacrein, which is a soluble organic matrix protein in the nacreous layer of oyster pearls. Northern blot analysis showed that the nacrein transcript was specifically expressed in mantle pallial. Analysis of the deduced amino acid sequence revealed that the protein contained two functional domains: one was a carbonic anhydrase and another was a Gly-Xaa-Asn (Xaa = Asp, Asn, or Glu) repeat domain; however, the carbonic anhydrase domain was split into two subdomains with insertion of the Gly-Xaa-Asn repeat domain between them. Our findings suggest that nacrein actually functions as a matrix protein whose repeated Gly-Xaa-Asn domain possibly binds calcium and as a carbonic anhydrase that catalyzes the HCO3- formation, thus participating in calcium carbonate crystal formation of the nacreous layer.

Molecular cloning and nucleotide sequencing of the Arthrobacter dextranase gene and its expression in Escherichia coli and Streptococcus sanguis.

A bacterial strain, which assimilated dextran and water-insoluble glucan produced by Streptococcus mutans, was isolated from soil. The bacterium produced and secreted potent dextranase activity, which was identified as Arthrobacter sp. and named CB-8. The dextranase was purified and some enzymatic properties were characterized. The enzyme efficiently decomposed the water-insoluble glucan as well as dextran. A gene library from the bacteria was constructed with Escherichia coli, using plasmid pUC19, and clones producing dextranase activity were selected. Based on the result of nucleotide sequencing analysis, it was deduced that the dextranase was synthesized in CB-8 cells as a polypeptide precursor consisting of 640 amino acid residues, including 49 N-terminal amino acid residues which could be regarded as a signal peptide. In the E. coli transformant, the dextranase activity was detected mostly in the periplasmic space. The gene for the dextranase was introduced into Streptococcus sanguis, using an E. coli-S. sanguis shuttle vector that contained the promoter sequence of a gene for glucosyltransferase derived from a strain of S. mutans. The active dextranase was also expressed and accumulated in S. sanguis cells.

Microbial production of pectin from citrus peel.

A new method for the production of pectin from citrus peel was developed. For this purpose, a microorganism which produces a protopectin-solubilizing enzyme was isolated and identified as a variety of Trichosporon penicillatum. The most suitable conditions for the pectin production were determined as follows. Citrus (Citrus unshiu) peel was suspended in water (1:2, wt/vol), the organism was added, and fermentation proceeded over 15 to 20 h at 30 degrees C. During the fermentation, the pectin in the peel was extracted almost completely without macerating the peel. By this method, 20 to 25 g of pectin was obtained per kg of peel. The pectin obtained was special in that it contained neutral sugar at high levels, which was determined to have a molecular weight suitable for practical applications.