Intracrystalline Proteins Research Articles

Intracrystalline proteins and calcium oxalate crystal degradation in MDCK II cells

We assessed the effects of intracrystalline urinary proteins on the ability of Type II Madin–Darby canine kidney (MDCK-II) cells to bind and degrade calcium oxalate monohydrate (COM) crystals. Binding of [ 14C]-labelled inorganic crystals (iCOM), and COM crystals precipitated from centrifuged and filtered (CF) or ultrafiltered (UF) human urine was quantified by radioactive analysis. SDS–PAGE confirmed the presence of intracrystalline proteins >10 kDa in CF crystals and their absence from UF crystals. Morphological effects were assessed qualitatively by field emission scanning electron microscopy. iCOM crystals bound rapidly and extensively and were resistant to degradation. Binding of CF crystals was weaker than UF crystals, and both had markedly less affinity than iCOM. CF and UF crystals were extensively degraded within 90 min, the effect being more pronounced with CF. These results support our hypothesis that intracrystalline proteins protect against urolithiasis by facilitating intracellular proteolytic digestion and destruction of crystals phagocytosed by urothelial cells.

Proteolysis and partial dissolution of calcium oxalate: a comparative, morphological study of urinary crystals from black and white subjects

Crystal adherence to the renal epithelium is widely regarded as a probable mechanism of stone formation. Intracrystalline proteins may provide access to the core of urinary crystals and thereby facilitate the dismantling of these crystals after their attachment to and phagocytosis by renal epithelial cells. The present study investigated the role of proteolysis and limited dissolution of urinary calcium oxalate (CaOx) crystals in South Africa's white and black populations with a view to understanding the remarkably low stone incidence in the black population compared with the whites. CaOx crystals were precipitated from filtered urine or ultrafiltered urine dosed with an intracrystalline protein, urinary prothrombin fragment 1 (UPTF1), isolated from white and black subjects. The crystals were fractured and subjected to proteolysis and/or limited dissolution before examination using field emission scanning electron microscopy (FESEM). FESEM data showed that CaOx crystals from white and black subjects were eroded by treatment with proteases. Cathepsin D caused the most significant crystal erosion, and more noticeable degradation of CaOx monohydrate (COM) crystals compared to CaOx dihydrate (COD). Limited dissolution studies showed the unique ultrastructures and fragmentation processes of COM and COD crystals. COM crystals appeared to be more susceptible to degradation and dissolution than CODs. Since COMs are predominant in blacks, compared with COD crystals from whites, it is speculated that the lower stone rate amongst South African blacks might be attributed partly to their more efficient destruction of retained COM crystals.

The nacre protein perlucin nucleates growth of calcium carbonate crystals.

Atomic force microscopy (AFM) in aqueous solution was used to investigate native nacre of the marine snail Haliotis laevigata on the microscopic scale and the interaction of purified nacre proteins with calcium carbonate crystals on the nanoscopic scale. These investigations were controlled by scanning electron microscopy (SEM), light microscopy (LM) and biochemical methods. For investigations with AFM and SEM, nacre was cleaved parallel to the aragonite tablets in this biogenic polymer/mineral composite. Multilamellar organic sheets consisting of a core of chitin with layers of proteins attached on both sides lay between the aragonite layers consisting of confluent aragonite tablets. Cleavage appeared to occur between the aragonite tablet layer and the protein layer. AFM images revealed a honeycomb-like structure to the organic material with a diameter of the 'honeycombs' equalling that of the aragonite tablets. The walls of the structures consisted of filaments, which were suggested to be collagen. The flat regions of the honeycomb-like structures exhibited a hole with a diameter of more than 100 nm. When incubated in saturated calcium carbonate solution, aragonite needles with perfect vertical orientation grew on the proteinacous surface. After treatment with proteinase K, no growth of orientated aragonite needles was detected. Direct AFM measurements on dissolving and growing calcite crystals revealed a surface structure with straight steps the number of which decreased with crystal growth. When the purified nacre protein perlucin was added to the growth solution (a super-saturated calcium carbonate solution) new layers were nucleated and the number of steps increased. Anion exchange chromatography of the water-soluble proteins revealed a mixture of about 10 different proteins. When this mixture was dialysed against saturated calcium carbonate solution and sodium chloride, calcium carbonate crystals precipitated together with perlucin leaving the other proteins in the supernatant. Thus perlucin was shown to be a protein able to nucleate calcium carbonate layers on calcite surfaces, and in the presence of sodium chloride, is incorporated as an intracrystalline protein into calcium carbonate crystals.

Selective inclusion of proteins into urinary calcium oxalate crystals: comparison between stone-prone and stone-free population groups

This study investigated whether incorporation of proteins into calcium oxalate urinary crystals is different in the black and white populations in South Africa and whether such differences could provide insight into the former group's remarkably low stone incidence. CaOx monohydrate (COM) and dihydrate (COD) crystals were precipitated from each group's urine after adjustment of the calcium concentrations to 0.5 and 12 mmol/l, respectively. Crystals were characterised by X-ray powder diffraction and scanning electron microscopy. Intracrystalline proteins were analysed by SDS-PAGE and immunodetected for urinary prothrombin fragment 1 (UPTF1) and osteopontin. Crystals precipitated from the black and white groups’ control urines comprised mainly COM and COD, respectively. In both race groups UPTF1 was the major protein included in pure COM crystals while in pure COD it was osteopontin, but in the black group osteopontin was also included in COM. The black group's urine crystals incorporated significantly more intracrystalline protein. Selective inclusion of UPTF1 and osteopontin may be due to the unique crystal structure of COM and COD and the proteins’ conformation at the different calcium concentrations at which these hydrates precipitate. The greater amount of intracrystalline inhibitory protein in the black group may be a factor in their low stone incidence.

Intracrystalline Proteins and Urolithiasis: A Synchrotron X-ray Diffraction Study of Calcium Oxalate Monohydrate

The existence of intracrystalline proteins and amino acids in calcium oxalate monohydrate was demonstrated by X-ray synchrotron diffraction studies. Their presence has implications for the destruction of calcium oxalate crystals formed in the urinary tract and the prevention of kidney stones. Although proteins are present in human kidney stones, their role in stone pathogenesis remains unknown. This investigation aimed to characterize the nature of the relationship between the organic and mineral phases in calcium oxalate monohydrate (COM) crystals grown in human urine and in aqueous solutions of proteins and amino acids to clarify the function of proteins in urolithiasis. COM crystals were grown in human urine and in aqueous solutions containing either human prothrombin (PT), Tamm-Horsfall glycoprotein (THG), aspartic acid (Asp), aspartic acid dimer (AspAsp), glutamic acid (Glu), glutamic acid dimer (GluGlu), or gamma-carboxyglutamic acid (Gla). Controls consisted of COM crystals precipitated from pure inorganic solutions or from human urine that had been ultrafiltered to remove macromolecules. Synchrotron X-ray diffraction with Rietveld whole-pattern peak fitting and profile analysis was used to determine nonuniform crystal strain and crystallite size in polycrystalline samples. Crystals precipitated from ultrafiltered urine had lower nonuniform strain than those grown in urine or in aqueous PT solution. Nonuniform strain was much lower in crystals grown in distilled water or in the presence of THG. For the amino acids, the highest nonuniform strain was exhibited by crystals grown in Gla solution, followed by Glu. Crystallite size was inversely related to nonuniform strain, with the effect being significantly less for amino acids than for macromolecules. Selected proteins and amino acids associated with COM crystals are intracrystalline. Although their incorporation into the mineral bulk would be expected to affect the rate of crystal growth, they also have the potential to influence the phagocytosis and intracellular destruction of any crystals nucleated and trapped within the renal collecting system. Crystals impregnated with protein would be more susceptible to digestion by cellular proteases, which would provide access to the crystal core, thereby facilitating further proteolytic degradation and mineral dissolution. We therefore propose that intracrystalline proteins may constitute a natural form of defense against renal stone formation.

Intracrystalline Proteins and the Hidden Ultrastructure of Calcium Oxalate Urinary Crystals: Implications for Kidney Stone Formation

Theexternal appearance of urinary calcium oxalate (CaOx) crystals suggests that they are solid, homogeneous structures, despite their known association with proteins. Our aim was to determine whether proteins comprising the organic matrix of CaOx crystals are superficial or intracrystalline in order to clarify the role of urinary proteins in the formation of kidney stones. CaOx crystals were precipitated from centrifuged and filtered, or ultrafiltered, healthy human urine. They were then treated with dilute NaOH to remove bound proteins, partially demineralized with EDTA, or fractured and subjected to limited proteolysis before examination by low-resolution scanning electron microscopy or field emission scanning electron microscopy. Crystals precipitated from centrifuged and filtered urine had a complex interior network of protein distributed throughout the mineral phase, which appeared to comprise closely packed subcrystalline particles stacked in an orderly array among an amorphous organic matrix. This ultrastructure was not evident in crystals deposited in the absence of macromolecules, which were completely solid. This is the first direct evidence that crystals generated from cell-free systems contain significant amounts of protein distributed throughout a complex internal cribriform ultrastructure. Combined with mineral erosion in the acidic lysosomal environment, proteins inside CaOx crystals would render them susceptible to attack by urinary and intracellular renal proteases and facilitate their further dissolution or disruption into small particles and ions for removal by exocytosis. The findings also have broader ramifications for industry and the materials sciences, as well as the development and resorption of crystals in biomineralization systems throughout nature.

A study of the correlation between organic matrices and nanocomposite materials in oyster shell formation

Calcium carbonate minerals are an integral part of many organisms. These biogenic minerals are all of original size, shape and high strength, and they are quite different from those found in their abiotic precipitates. It has been accepted that the formation, morphological development and crystallography of the biocomposites are controlled by the intrinsic molecular recognition of macromolecules. In this study, with the analyses of X-ray diffraction and scanning electron microscopy of the texture of biogenic minerals in oyster shells, we noted that the intracrystalline proteins deliberately reduce the coherence lengths of biogenic crystals compared to synthetic ones, leading to more isotropy. In order to understand the exact nature of the controlled nucleation and growth, we investigated the changes in protein conformation in vivo from the mineral-specific layers using Fourier self-deconvolution and Gaussian curve-fitting techniques. And via in vitro assays, we studied the relation of such changes to biomineral phase and morphology. We showed that the shell proteins in vivo are in the higher structural ordered state, and β-antiparallel structure was predominant in each shell layer. Also, as the shell undergoes a change of calcium carbonate polymorphs from aragonite to calcite, significant alterations of the protein conformation with the denaturing of α-helix and β-structure in the aragonitic layer is induced. These results provide a relationship between the effects of conformational changes on the nanostructure of biocomposites and the necessity of new synthetic strategies.

Erratum: ?Model peptide studies of sequence repeats derived from the intracrystalline biomineralization protein, SM50. I. GVGGR and GMGGQ repeats?, Volume 49, No. 4, pp. 303-312 (1999)

Erratum: ?Model peptide studies of sequence repeats derived from the intracrystalline biomineralization protein, SM50. I. GVGGR and GMGGQ repeats?, Volume 49, No. 4, pp. 303-312 (1999)

Degradation of intracrystalline proteins and amino acids in fossil brachiopods

Four genera of Recent to Plio-Pleistocene articulated brachiopods were collected from up to 16 horizons spanning the last 3.3 Ma of sediment deposition in the South Wanganui Basin, New Zealand, and assayed for the preservation of intracrystalline proteins and/or amino acids. The proteins present in the shells of living and Recent brachiopods undergo rapid degradation through the decomposition of the peptide bond. Up to 95% of the constituent amino acids from the proteins are present in the free state by 0.12 Ma. This rate of degradation is far higher than was originally expected for intracrystalline proteins. Quantitative analysis of the concentrations of amino acids present within the shells of fossil brachiopods indicates a range of reaction rates for the subsequent degradation of individual amino acids. The degradation of these amino acids may lead to the total loss of compounds, to the generation of non-standard amino acids, or to diagenetically produced proteinaceous amino acids. These reactions do not necessarily mirror those which occur during the pyrolysis of an aqueous solution of the pure amino acids, either in their rate or products.

Morphogenesis of calcitic sponge spicules: a role for specialized proteins interacting with growing crystals

Crystals formed in biological tissues often adopt remarkable morphologies that are thought to be determined mainly by the shapes of the confined spaces in which they grow. Another possible way of controlling crystal shape, demonstrated only in vitro, is by means of specialized proteins preferentially interacting with certain crystal faces. In so doing, they reduce the rate of growth in these directions and consequently change the overall crystal shape. In an X-ray diffraction study of the distribution of defects within the lattice of calcite crystals produced by certain sponges, we show that a remarkable correlation exists between the defect patterns or crystal texture and the macroscopic morphology of the spicules. This was observed in two cases in which proteins are present within the spicule crystal, but not in a third case where such intracrystalline proteins are absent. Furthermore, one of the spicules exhibited marked differences in texture even within families of structurally identical crystal planes, demonstrating that the organisms exert exquisite control over the microenvironment in which crystals grow. We conclude that highly controlled intercalation of specialized proteins inside the crystals is an additional means by which organisms control spicule growth.

Controlled Occlusion of Proteins: A Tool for Modulating the Properties of Skeletal Elements

Abstract Composite materials in which the organic host is stiffened by guest particles, are widely used in nature and are produced synthetically by man. Organisms also produce a different type of composite in which the host is a crystal and the guests are macromolecules occluded in an orderly fashion within the crystal. The best studied examples, to date, are biogenic calcite crystals, and in particular those formed by the echinoderms. In vitro experiments with calcite crystals grown in the presence of echnioderm intracrystalline proteins, show that these macromolecules are occluded inside the crystal on specific planes, and their presence alters the mechanical properties of the crystal host. Furthermore, the proteins also influence the crystal textural properties. Model studies using crystals of dicarboxylic acid salts grown in the presence of intracrystalline proteins show that the coherence length is reduced in directions perpendicular to the planes on which the proteins adsorb. We found anisotropic effects in almost all the biogenic calcite crystals we examined. Furthermore, we noted an interesting relationship between the variations in coherence length in the different crystallographic directions and the gross morphology of the single crystal elements, suggesting that these proteins may also function in determining the morphology of the crystal during growth. These novel single crystalprotein composites may be just one example of strategies used in nature for producing materials with special properties.

Biogeochemistry of brachiopod intracrystalline proteins and amino acids

Brachiopods contain several classes of intracrystalline molecules, secreted during the growth of the organism, and subsequently incorporated in the shell during biomineralization. The function of these molecules is not clear, although in the Mollusca, molecules in similar sites are thought to be involved with biologically controlled mineralization. The order of amino acids, and hence the quantity of each amino acid in proteins is determined by the genome of the organism, and study of such molecules should reveal information regarding the genomes of the fossil organisms.Previous studies of fossil molecules from brachiopods have concentrated on the web of intracrystalline molecules which surrounds the crystallites of the secondary fibres of the shell. Work with Recent articulate brachiopods has shown that this web decays in less than a year, and hence any remnants of this web in the fossil record are likely to be highly degraded and unrepresentative of the original composition of the protein.In contrast to this, intracrystalline molecules will be protected, encased within the calcium carbonate of the shell and analogous in many ways to fluid inclusions found in inorganic minerals. In such sites, contaminant and degradative factors such as microorganisms and large concentrations of water will be excluded and the effects of the enclosing sediments negated. Any breakdown of the molecules will be in situ, and the detectable products analysed.In this study, intracrystalline molecules have been extracted from the shells of Recent and fossil brachiopods from the Plio-Pleistocene South Wanganui Basin, New Zealand. The molecules were analysed for free and peptide bound amino acids from both the soluble and insoluble fractions. The state of preservation of the molecules and their taxonomic specifity were analysed.It was found that, even in these protected sites, individual amino acids were up to 90% in the free state i.e. that the proteins had mostly been hydrolyzed by the action of time and any water remaining in the shell structure after 0.12 Ma. Amino acids which are particularly sensitive to degradative reactions, such as serine and threonine were totally lost from the soluble fraction. However, the use of multivariate statistical analyses on data from the soluble remains of the protein indicates that decay does not render the molecules useless for taxonomy; groupings can be made on the basis of analyses alone.Progressively older samples tend to have lower yields of amino acid in the soluble fraction, and this tends to correspond with an increase in the relative proportion of intracrystalline insoluble compounds. Although there is no direct linear relationship between the age of a sample and the proportion of insoluble compounds, older samples tend to have more insolubles. It is clear that other factors must play a part in the formation of these insoluble compounds. Preliminary studies of their amino acid composition indicates the presence of sensitive amino acids such as serine, indicating the stabilising effects of these diagenetic compounds.The degradation of amino acids follows complex decay pathways, with some decaying to non-amino compounds and others to other amino acids, the most common being alanine from the dehydration of serine. The increasing concentrations of these amino acids can be a distorting factor in molecular taxonomy from fossil molecules.ACKNOWLEDGEMENTS. This work was completed during the tenure of a UK NERC studentship to DW (GT4/89/GS/42) and funding from the Royal Society to GBC.

Interactions of sea-urchin skeleton macromolecules with growing calcite crystals— a study of intracrystalline proteins

The exoskeletons of sea urchins are composed of magnesiumbearing calcite. Individual test plates and spines behave as single crystals in polarized light or when examined by X-ray diffraction1–3. They do not, however, cleave like inorganic calcite crystals along the {104} hexagonal cleavage planes, but have conchoidal fracture surfaces reminiscent of amorphous glass. Discussion of this paradox revolves around whether the phase is monocrystalline2,4,5, multicrystalline6, or some combination thereof7, but provides no explanation for the phenomenon. To address this question we grew crystals of calcite in the presence of acidic glycoproteins extracted from within the mineralized hard parts of sea-urchin tests8–10. As a control we used analogous proteins from the calcitic layer of a mollusc shell which are known to be nucleators of calcite when adsorbed on a rigid substrate, but inhibitors when in solution11–13. We show that the sea urchin, but not the mollusc macromolecules selectively adsorb onto specific calcite crystal planes and with continued crystal growth are occluded inside the solid phase. These synthetic crystals fracture with a conchoidal cleavage similar to that observed in sea-urchin calcite. Thus intracrystalline proteins may be responsible for this phenomenon in biology and the manner in which they affect the mechanical properties of the crystals may also have interesting implications to the materials sciences.