Ethylenediamine-tetraacetic acid extracted water-soluble matrix proteins in molluscan shells secreted from the mantle epithelia are believed to control crystal nucleation, morphology, orientation, and phase of the deposited mineral. Previously, atomic force microscopy demonstrated that abalone nacre proteins bind to growing step edges and to specific crystallographic faces of calcite, suggesting that inhibition of calcite growth may be one of the molecular processes required for growth of the less thermodynamically stable aragonite phase. Previous experiments were done with protein mixtures. To elucidate the role of single proteins, we have characterized two proteins isolated from the aragonitic component of nacre of the red abalone, Haliotis rufescens. These proteins, purified by hydrophobic interaction chromatography, are designated AP7 and AP24 (aragonitic protein of molecular weight 7 kDa and 24 kDa, respectively). Degenerate oligonucleotide primers corresponding to N-terminal and internal peptide sequences were used to amplify cDNA clones by a polymerase chain reaction from a mantle cDNA library; the deduced primary amino acid sequences are presented. Preliminary crystal growth experiments demonstrate that protein fractions enriched in AP7 and AP24 produced CaCO(3) crystals with morphology distinct from crystals grown in the presence of the total mixture of soluble aragonite-specific proteins. Peptides corresponding to the first 30 residues of the N-terminal sequences of both AP7 and AP24 were generated. The synthetic peptides frustrate the progression of step edges of a growing calcite surface, indicating that sequence features within the N-termini of AP7 and AP24 include domains that interact with CaCO(3). CD analyses demonstrate that the N-terminal peptide sequences do not possess significant percentages of alpha-helix or beta-strand secondary structure in solution. Instead, in both the presence and absence of Ca(II), the peptides retain unfolded conformations that may facilitate protein-mineral interaction.

Gaucher disease results from mutations in the lysosomal enzyme acid beta-glucosidase (GCase). Although enzyme replacement therapy has improved the health of some affected individuals, such as those with the prevalent N370S mutation, oral treatment with pharmacological chaperones may be therapeutic in a wider range of tissue compartments by restoring sufficient activity of endogenous mutant GCase. Here we demonstrate that isofagomine (IFG, 1) binds to the GCase active site, and both increases GCase activity in cell lysates and restores lysosomal trafficking in cells containing N370S mutant GCase. We also compare the crystal structures of IFG-bound GCase at low pH with those of glycerol-bound GCase at low pH and apo-GCase at neutral pH. Our data indicate that IFG induces active GCase, which is secured by interactions with Asn370. The design of small molecules that stabilize substrate-bound conformations of mutant proteins may be a general therapeutic strategy for diseases caused by protein misfolding and mistrafficking.

Gaucher disease is a lysosomal storage disorder caused by deficiency in lysosomal acid ␤-glucosidase (GlcCerase), the enzyme responsible for the catabolism of glucosylceramide. One of the most prevalent disease-causing mutations, N370S, results in an enzyme with lower catalytic activity and impaired exit from the endoplasmic reticulum. Here, we report that the iminosugar isofagomine (IFG), an active-site inhibitor, increases GlcCerase activity 3.0 ؎ 0.6-fold in N370S fibroblasts by several mechanisms. A major effect of IFG is to facilitate the folding and transport of newly synthesized GlcCerase in the endoplasmic reticulum, thereby increasing the lysosomal pool of the enzyme. In addition, N370S GlcCerase synthesized in the presence of IFG exhibits a shift in pH optimum from 6.4 to 5.2 and altered sensitivity to SDS. Although IFG fully inhibits GlcCerase in the lysosome in an in situ assay, washout of the drug leads to partial recovery of GlcCerase activity within 4 h and full recovery by 24 h. These findings provide support for the possible use of active-site inhibitors in the treatment of some forms of Gaucher disease.

Extracellular adhesives from the diatoms Achnanthes longipes, Amphora coffeaeformis, Cymbella cisfula, and Cymbella mexicana were characterized by monosaccharide and methylation analysis, lectin-fluorescein isothiocyanate localization, and cytochemical staining. Polysaccharide was the major component of adhesives formed during cell motility, synthesis of a basal pad, and/or production of a highly organized shaft. Hot water-insoluble/hot 0.5 M NaHC0,-soluble anionic polysaccharides from A. longipes and A. coffeaeformis adhesives were primarily composed of galactosyl (64-70%) and fucosyl (32-42%) residues. I n A. longipes polymers, 2,3-, f-, 3-, and 4-linked/substituted galactosyl, f-, 3-, 4-, and 2-linked fucosyl, and t-and 2-linked glucuronic acid residues predominated. Adhesive polysaccharides from C. cistula were EDTAsoluble, sulfated, consisted of 83% galactosyl (4-, 4,6-, and 3,4-linked/substituted) and 13% xylosyl (t-, 4,/5,-, and 3,-linked/ substituted) residues, and contained no uronosyl residues. Ulex europaeus agglutinin uniformly localized cu(l,2)-~-fucose units in C. cistula and Achnanthes adhesives formed during motility and in the pads of A. longipes. D-Calactose residues were localized throughout the shafts of C. cisfula and capsules of A. coffeaeformis. D-Mannose and/or o-glucose, D-galactose, and a(t)-i-fucose residues were uniformly localized in the outer layers of A. longipes shafts by Cancavalia ensiformis, Abrus precatorim, and Lotus tefragonolobus agglutinin, respectively. A model for diatom cell adhesive structure was developed from chemical characterization, localization, and microscopic observation of extracellular adhesive components formed during the diatom cell-attachment process.Achnanthes longipes, Cymbella cistula, Cymbella mexicana, and Amphora cofeaeformis are relatively large, fast-growing, unicellular organisms specialized in cell motility and attachment via distinct extracellular structures. These adhesive structures can be easily manipulated, observed, and isolated for the study of synthesis, transport, modification, and assembly of extracellular polymers. Modeling such systems can provide a better understanding of how plant cells adhere to surfaces and interact with their surrounding environments. Furthermore, these diatoms are a major component of marine and freshwater biofilms (Round et al., 1990) and thus cause a variety of biofouling problems (Alberte et al., 1992). Understanding how these cells adhere to surfaces will also aid in the development of new antibiofouling surfaces and of adhesives for use in marine and freshwater environments.Most diatoms attach to surfaces via polymers excreted from a slit (raphe) or apical pore field in the siliceous cell wall (frustule) (Hoagland et al., 1993). Exuded polymers are assembled into a variety of structures, such as trails (material left behind during motility), sheaths (organic matrices tightly associated with the cell wall), capsules (organic matrices loosely associated with the cell walls), and stalks (permanent attachment s...

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene encoding alpha-galactosidase A (alpha-Gal A), with consequent accumulation of its major glycosphingolipid substrate, globotriaosylceramide (GL-3). Over 500 Fabry mutations have been reported; approximately 60% are missense. The iminosugar 1-deoxygalactonojirimycin (DGJ, migalastat hydrochloride, AT1001) is a pharmacological chaperone that selectively binds alpha-Gal A, increasing physical stability, lysosomal trafficking, and cellular activity. To identify DGJ-responsive mutant forms of alpha-Gal A, the effect of DGJ incubation on alpha-Gal A levels was assessed in cultured lymphoblasts from males with Fabry disease representing 75 different missense mutations, one insertion, and one splice-site mutation. Baseline alpha-Gal A levels ranged from 0 to 52% of normal. Increases in alpha-Gal A levels (1.5- to 28-fold) after continuous DGJ incubation for 5 days were seen for 49 different missense mutant forms with varying EC(50) values (820 nmol/L to >1 mmol/L). Amino acid substitutions in responsive forms were located throughout both structural domains of the enzyme. Half of the missense mutant forms associated with classic (early-onset) Fabry disease and a majority (90%) associated with later-onset Fabry disease were responsive. In cultured fibroblasts from males with Fabry disease, the responses to DGJ were comparable to those of lymphoblasts with the same mutation. Importantly, elevated GL-3 levels in responsive Fabry fibroblasts were reduced after DGJ incubation, indicating that increased mutant alpha-Gal A levels can reduce accumulated substrate. These data indicate that DGJ merits further evaluation as a treatment for patients with Fabry disease with various missense mutations.

The lustrin protein superfamily represents a class of elastomeric biomineralization proteins which are localized between layered aragonite mineral plates (i.e., nacre layers) in mollusk shell. These proteins exhibit adhesive behavior within the mineralized matrix, suggesting that there may be interfacial interactions between lustrin proteins and aragonite mineral surfaces and/or other surfaces of organic matrix components. Previously, within one lustrin protein (Lustrin A) we identified a 24 amino acid Asp-rich polyelectrolyte sequence, named D4, which adopts an open, unfolded conformation in solution. This sequence contains a putative Ca(II) interaction sequence, −DTDADSGSD−, which potentially represents a site for Ca(II)-mediated Lustrin A−mineral or Lustrin A−organic matrix interactions. In this present study, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, circular dichroism, and nuclear magnetic resonance spectroscopy, we confirm that the D4 sequence is a Ca(II)-binding polypeptide that maintains its unfolded conformation in the presence of Ca(II) ions. Moreover, using geological calcite overgrowth assays and scanning electron microscopy visualization, we observe interruption in calcium carbonate crystal growth in the presence of nanomolar levels of D4 polypeptide. Proton solid-state nuclear magnetic resonance experiments verify that during the course of calcium carbonate crystal growth D4 becomes bound to calcite fragments and cannot be entirely displaced from the mineral fragments using low and high ionic strength conditions. We conclude from our studies that D4 represents a potential matrix adhesion site within the Lustrin A protein and possesses molecular traits that coincide with other reported calcium carbonate mineral interaction domains.

Lysosomal enzymes are responsible for the degradation of a wide variety of glycolipids, oligosaccharides, proteins, and glycoproteins. Inherited mutations in the genes that encode these proteins can lead to reduced stability of newly synthesized lysosomal enzymes. While often catalytically competent, the mutated enzymes are unable to efficiently pass the quality control mechanisms of the endoplasmic reticulum, resulting in reduced lysosomal trafficking, substrate accumulation, and cellular dysfunction. Pharmacological chaperones (PCs) are small molecules that bind and stabilize mutant lysosomal enzymes, thereby allowing proper cellular translocation. Such compounds have been shown to increase enzyme activity and reduce substrate burden in a number of preclinical models and clinical studies. In this Perspective, we review several of the lysosomal diseases for which PCs have been studied and the SAR of the various classes of molecules.

Gaucher disease is caused by mutations in the gene that encodes the lysosomal enzyme acid b-glucosidase (GCase). We have shown previously that the small molecule pharmacological chaperone isofagomine (IFG) binds and stabilizes N370S GCase, resulting in increased lysosomal trafficking and cellular activity. In this study, we investigated the effect of IFG on L444P GCase. Incubation of Gaucher patient-derived lymphoblastoid cell lines (LCLs) or fibroblasts with IFG led to approximately 3.5-and 1.3-fold increases in L444P GCase activity, respectively, as measured in cell lysates. The effect in fibroblasts was increased approximately 2-fold using glycoprotein-enrichment, GCase-immunocapture, or by incubating cells overnight in IFG-free media prior to assay, methods designed to maximize GCase activity by reducing IFG carryover and inhibition in the enzymatic assay. IFG incubation also increased the lysosomal trafficking and in situ activity of L444P GCase in intact cells, as measured by reduction in endogenous glucosylceramide levels. Importantly, this reduction was seen only following three-day incubation in IFG-free media, underscoring the importance of IFG removal to restore lysosomal GCase activity. In mice expressing murine L444P GCase, oral administration of IFG resulted in significant increases (2-to 5-fold) in GCase activity in disease-relevant tissues, including brain. Additionally, eight-week IFG administration significantly lowered plasma chitin III and IgG levels, and 24-week administration significantly reduced spleen and liver weights. Taken together, these data suggest that IFG can increase the lysosomal activity of L444P GCase in cells and tissues. Moreover, IFG is orally available and distributes into multiple tissues, including brain, and may thus merit therapeutic evaluation for patients with neuronopathic and non-neuronopathic Gaucher disease.