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At least 19 recordsLinked to original sources
PubMed1996

Pancreatic lithostathine as a calcite habit modifier.

Most biological fluids are supersaturated with calcium salts. A mechanism controlling crystal growth is therefore necessary to prevent excessive precipitation and development of a lithiasis. In pancreatic juice, calcite precipitation is prevented by lithostathine, a glycoprotein that inhibits calcite crystal growth. We describe here the interaction of lithostathine with calcite crystals. Without lithostathine, calcite crystals grew as rhombohedra showing six (104) faces. At low concentration (1 microM), lithostathine already altered crystal growth by generating new (110) faces. At physiological concentrations (3-10 microM), adsorption resulted in a transition from rhombohedral to sub-cubic habits. Immunochemical localization demonstrated that, although all (104) faces are equivalent, lithostathine binding was restricted to the face edges distal to the c axis. Scanning electron microscopy showed that, at the site of lithostathine binding, spreading of new CaCO3 layers during crystal growth was arrested before reaching the crystal diad axis-bearing edges. The successive kinks generated during crystal growth formed the new, striated (110)faces. Similar modifications were observed with the N-terminal undecapeptide of lithostathine that bears the inhibitory activity. With 100 microM lithostathine, (110) faces could reach the c axis outcrop of the former rhombohedron, resulting in an olive-shaped crystal. Finally, the number of crystals increased and their average size decreased when lithostathine concentration increased from 0.1 to 100 microM. Decreased Ca2+ concentration during crystal growth was delayed in the presence of lithostathine. It was concluded that lithostathine controls lithogenesis 1) by triggering germination of numerous calcite crystals and 2) by inhibiting the rate of Ca2+ ion apposition on the nuclei and therefore interfering with the apposition of new layers on calcite. Formation of smaller crystals, whose elimination is easier, is thereby favored.

Calcium Carbonate↗
PubMed1997

Pancreatic stone protein (lithostathine), a physiologically relevant pancreatic calcium carbonate crystal inhibitor?

Apart from digestive enzymes, pancreatic juice contains several proteins that are not directly involved in digestion. One of these, lithostathine, has been reported to exhibit calcite crystal inhibitor activity in vitro. As pancreatic juice is supersaturated with respect to calcium carbonate, it was hypothesized that lithostathine stabilizes pancreatic juice. Lithostathine is cleaved by trace amounts of trypsin, resulting in a C-terminal polypeptide and an N-terminal undecapeptide, which has been identified as the active site of lithostathine regarding crystal inhibition. We produced rat lithostathine in a baculovirus expression system. In order to test its functional activity, the protein was purified using a nondenaturing multi-step procedure. In the low micromolar range, recombinant rat lithostathine in vitro exhibited calcite crystal inhibitor activity, confirming earlier reports. Limited tryptic proteolysis of recombinant lithostathine was performed, and the two cleavage products were separated; the C-terminal polypeptide was precipitated by centrifugation, and the N-terminal undecapeptide was purified by high performance liquid chromatography. Only the C-terminal peptide displayed measurable calcite crystal inhibitory activity. Furthermore, synthetic undecapeptides with identical sequence to the N-terminal undecapeptides of rat or human lithostathine were inactive. However, when tested in the same in vitro assays, other pancreatic or extra-pancreatic proteins show inhibitory activity in the same concentration range as lithostathine, and inorganic phosphate is active as well. Based on these findings it seems unlikely that lithostathine is a physiologically relevant calcite crystal inhibitor. The name "lithostathine" is therefore inappropriate, and the protein's key function remains to be elucidated.

Amino Acid Sequence↗
PubMed1998

Quantification of human lithostathine S2-5 forms using the antibody to the N-terminal peptide region.

Lithostathine S2-5 inhibits in vitro crystal growth of CaCO3. We developed an antibody against the peptide region responsible for inhibitory effect to determine whether lithostathine S2-5 levels are different in the pancreatic juice of patients with and without chronic pancreatitis. The antibody against the synthetic peptide of the N-terminal end of lithostathine S2-5 detected lithostathine S2-5 but not lithostathine S1 or lithostathine extracted from pancreatic calculi. Lithostathine S2-5 was detected in samples of pancreatic juice protein by immunoblotting using the specific antibody. The concentration of lithostathine S2-5 was compared between control and chronic pancreatitis groups. The mean concentrations of lithostathine S2-5 were significantly (p=0.002) lower in chronic pancreatitis, 16.3 microg/mg of total protein, than in the control, 47.1 microg/mg of total protein. A decreased concentration of lithostathine S2-5 seems to increase the risk of stone formation in the ducts during the course of chronic pancreatitis because of insufficient inhibition of CaCO3 crystal growth.

Adult↗
PubMed1995

Purification and assay of secretory lithostathine in human pancreatic juice by fast protein liquid chromatography.

Impaired secretion of lithostathine, a pancreatic glycoprotein capable of inhibiting the growth of CaCO3 crystals, has been reported in chronic calcifying pancreatitis. Controversial results were obtained, however, using immunoassays with different antibodies. The aim of this study was to purify and to measure juice lithostathine by a non-immunological method. Fast protein liquid chromatography (FPLC) on a cation exchange column eluted by a sodium chloride gradient, was used. The conditions appropriate to separate secretory (S) from hydrolysed (H) isoforms of immunopurified lithostathine were also used for juice analysis. Pancreatic juice was collected by endoscopic cannulation of the major pancreatic duct, after secretin stimulation, from eight patients with chronic pancreatitis (CP) and from eight controls. In all samples, S-isoforms of lithostathine (ranging from 16 to 19 Mr at SDS-PAGE) were the only constituent of two of the 15 peaks in which FPLC resolved the pancreatic proteins. The nature of these two peaks was confirmed by their coelution with immunopurified S-lithostathine and by immunoblot analysis with polyclonal anti-lithostathine antibodies. The ratio between the area of S-lithostathine peaks and the total area of proteic eluates, was always lower in CP patients (5.3 micrograms/mg of protein, median value; 0.2-15.4, range) than in controls (35.2 micrograms/mg; 16.6-55.9). It is concluded that lithostathine can be purified and measured in pancreatic juice by FPLC. Our results with a nonimmunological assay confirm a reduced secretion of lithostathine in patients with CP.

Adult↗
PubMed1998

Lithostathine, the presumed pancreatic stone inhibitor, does not interact specifically with calcium carbonate crystals.

Lithostathine (pancreatic stone protein, Reg protein) is, in addition to albumin, the major nonenzymatic protein of the pancreatic juice. It has been assumed to inhibit calcium carbonate precipitation and therefore to prevent stone formation in the pancreatic ducts. This function is, however, debatable. The assumption is based on the inhibition of in vitro crystal nucleation and growth by lithostathine. Considering that these phenomena occur only under certain critical conditions, we re-examined the question using a protein preparation where the purity and folding have been tested by mass spectroscopy and NMR in the absence of nonprotein contaminants. Under these conditions, we showed conclusively that lithostathine does not inhibit calcium carbonate nucleation and crystal growth. We demonstrated that previous findings on the alleged inhibition can be attributed to the uncontrolled presence of salts in the protein preparation used. Moreover, the affinity of lithostathine to calcite crystals, expressed as the half-life of bound iodinated protein in the presence of unlabeled competitor, was significantly lower than that of bovine serum albumin (8.8 and 11.2 h, respectively). Using glass microspheres instead of crystals did not significantly change the half-life of bound lithostathine (8.0 h). These findings are incompatible with the hypothesis of a specific interaction of lithostathine with calcium carbonate crystals. In conclusion, considering that components of pancreatic juice such as NaCl and phosphate ions are powerful inhibitors of calcium carbonate crystal growth, the mechanism of stone formation in pancreatic ducts must be reconsidered. The presence in normal pancreatic juice of small amounts of the 133-residue isoform of lithostathine (PSP-S1), which precipitates at physiological pH, should be noted, and the possibility should be considered that they form micro-precipitates that aggregate and are progressively calcified.

Calcium↗
PubMed1995

Quantification of human lithostathine by high performance liquid chromatography.

Pancreatic stones of patients with chronic calcifying pancreatitis (CCP) are mostly made up of CaCO3 crystals. Formation and growth of such crystals is inhibited in vitro by lithostathine, a protein present in normal pancreatic juice. Decreased lithostathine activity was therefore suspected in patients with CCP, but comparison by immunoassay of lithostathine concentrations in the pancreatic juices of patients and controls led to conflicting results. This study shows that these discrepancies might have been caused in part by a remarkably high susceptibility of the protein to trypsin like cleavage, resulting in important structural changes and concomitant modifications of the epitopes. A novel lithostathine assay in juice was developed, based on separation of secretory proteins by high performance liquid chromatography. The chromatographic separation of lithostathine was based on hydrophobic interactions at pH 5.0 using a Phenyl-TSK column. This study showed with this assay that lithostathine concentrations (microgram/mg of total protein) were similar in CCP patients with alcoholic aetiology (mean (SD) 6.3 (2.7)) and other aetiologies (7.2 (3.7)), but one third of those estimated in patients without pancreatic disease (16.7 (4.3)). Similar concentrations were found, however, in chronic alcoholic patients without CCP (6.6 (3.3)) and in patients with CCP. It was concluded that decreased lithostathine concentration is associated with CCP, although such a decrease is not sufficient by itself for the disease to occur.

Alcoholism↗
PubMed1993

[Pancreatic lithostathine inhibitor of calcium carbonate precipitation: structure-function relationship].

Pancreatic juice is naturally supersatured in calcium and bicarbonate ions. A mechanism controlling CaCO3 crystal formation and growth is therefore necessary to prevent duct clogging. Lithostathine, a glycoprotein synthesized by acinar cells and secreted in pancreatic juice, could be involved in such a control. Lithostathine significantly delayed crystal nucleation and inhibited growth of CaCO3 crystals from supersatured solutions. Lithostathine adsorbed to sites specifically inhibiting crystal growth with a dissociation constant Kd = 0.9 x 10(-6) mol/L. The glycosylated N-terminal undecapeptide generated by limited trypsin hydrolysis of lithostathine, inhibited CaCO3 crystal growth with a Kd = 3.4 x 10(-6) mol/L similar to that of lithostathine. On the contrary, the carboxy-terminal polypeptide (lithostathine H) was inactive. The N-terminal undecapeptide of lithostathine is therefore essential to the inhibitory activity of the protein on CaCO3 crystal growth.

Adsorption↗
PubMed1992

Inhibition of nucleation and crystal growth of calcium carbonate by human lithostathine.

Pancreatic juice is naturally supersaturated in calcium and bicarbonate ions. A mechanism controlling CaCO3 crystal formation and growth is therefore necessary to prevent duct clogging. The present study shows that lithostathine, a glycoprotein present in human pancreatic juice at a concentration in the range of 10 mumol/L, could be involved in such a control. Lithostathine in concentrations greater than 1.5 mumol/L significantly delayed crystal nucleation and inhibited growth of preformed CaCO3 crystals from supersaturated solutions. Adsorption of lithostathine on crystals was shown by immunodetection. Albumin also adsorbed on CaCO3 crystals, but neither albumin nor other pancreatic secretory proteins inhibited crystal nucleation or growth. Lithostathine adsorbed to sites specifically inhibiting crystal growth with a dissociation constant (Kd) = 0.9 x 10(-6) mol/L. The glycosylated amino-terminal undecapeptide generated by limited trypsin hydrolysis inhibited CaCO3 crystal growth with a Kd = 3.0 x 10(-6) mol/L, similar to that of lithostathine. On the contrary, the carboxy-terminal polypeptide was inactive. A synthetic undecapeptide identical to the N-terminal end but not glycosylated was equally active. The activity disappeared upon digestion of the undecapeptide with V8 protease. The N-terminal undecapeptide of lithostathine is therefore essential to the inhibitory activity of the protein on CaCO3 crystal growth.

Adsorption↗
PubMedDate not supplied

Lithostathine and pancreatitis-associated protein are involved in the very early stages of Alzheimer's disease.

According to one of the theories formulated to explain the etiology of Alzheimer's disease (AD), amylosis may reflect a specific inflammatory response. Two inflammatory proteins, lithostathine and PAP, were evidenced by immunohistochemistry in senile plaques and neurofibrillary tangles of patients with AD. In addition, lithostathine and PAP were significantly increased in the cerebrospinal fluid of patients with AD when compared to patients with multiple sclerosis, another inflammatory disease, and to normal control subjects. However, no correlation was observed with age of occurrence. Furthermore, lithostathine and PAP were increased even at the very early stages of AD, and their level remained elevated during the course of the AD unlike TNFalpha whose level, very high at very early stages, regularly decreased. Finally, if part of lithostathine and PAP are synthesized in the brain, a large part comes from serum by passage over the blood-brain barrier. These results indicate (i) the existence of an acute phase response followed by a chronic inflammation in AD, and (ii) that lithostathine and PAP are involved even at the first pre-clinical biochemical events of AD. In addition, because lithostathine undergoes an autolytic cleavage leading to its precipitation and the formation of fibrils, we believe that it may be involved in amyloidosis and tangles by allowing heterogeneous precipitation of other proteins.

Acute-Phase Proteins↗
PubMed1998

Lithostathine messenger RNA expression in different types of chronic pancreatitis.

Lithostathine may play a physiological role in preventing the precipitation of excess calcium in the pancreatic juice. The hypothesis has been advanced that in chronic calcifying pancreatitis the abnormal biosynthesis of lithostathine might be the original defect to which genetic proneness to the disease may be ascribed. The aim of the present work was to study lithostathine messenger RNA expression in the pancreas of patients with different types of pancreatitis. Lithostathine and chymotrypsinogen mRNA were determined in surgical specimens obtained from the pancreases of the following subjects: (a) 13 patients with chronic alcoholic pancreatitis (84.6% calcified); (b) 4 patients with chronic hereditary pancreatitis (all calcified); (c) 6 patients with chronic obstructive pancreatitis (4 calcified); and (d) 27 subjects suffering from pancreatic cancer. Significantly lower concentrations of both mRNAs were found in the pancreases of chronic pancreatitis patients than in non-cancerous tissue from pancreatic cancer subjects. However, about 70% of the pancreatic cancer subjects showed lithostathine and chymotrypsinogen mRNA levels comparable to those of chronic pancreatitis patients. These results indicate that the decrease in the level of mRNA is not specific to lithostathine and it is unrelated to the presence of pancreatic stones.

Adult↗
PubMed1999

Biophysical characterization of lithostathine. Evidences for a polymeric structure at physiological pH and a proteolysis mechanism leading to the formation of fibrils.

Lithostathine is a calcium carbonate crystal habit modifier. It is found precipitated under the form of fibrils in chronic calcifying pancreatitis or Alzheimer's disease. In order to gain better insight into the nature and the formation of fibrils, we have expressed and purified recombinant lithostathine. Analytical ultracentrifugation and quasi-elastic light scattering techniques were used to demonstrate that lithostathine remains essentially monomeric at acidic pH while it aggregates at physiological pH. Analysis of these aggregates by electron microscopy showed an apparently unorganized structure of numerous monomers which tend to precipitate forming regular unbranched fibrils. Aggregated forms seem to occur prior to the apparition of fibrils. In addition, we have demonstrated that these fibrils resulted from a proteolysis mechanism due to a specific cleavage of the Arg(11)-Ile(12) peptide bond. It is deduced that the NH(2)-terminal undecapeptide of lithostathine normally impedes fiber formation but not aggregation. A theoretical model explaining the formation of amyloid plaques in neurodegenerative diseases or stones in lithiasis starting from lithostathine is described. Therefore we propose that lithostathine, whose major function is unknown, defines a new class of molecules which is activated by proteolysis and is not involved in cytoskeleton nor intermediate filament functions.

Alzheimer Disease↗
PubMed1996

Both ethanol and protein deficiency increase messenger RNA levels for pancreatic lithostathine.

Both ethanol abuse and protein deficiency are well known associations of chronic pancreatitis. An early event in chronic pancreatitis is the deposition of protein plugs in small pancreatic ducts, leading to ductular obstruction and acinar cell damage. Lithostathine, a pancreatic secretory protein, is a major organic component of protein plugs. The aim of this study was to determine the effect of chronic ethanol administration and dietary protein deficiency, separately and in combination, on messenger RNA (mRNA) levels for pancreatic lithostathine. Male Sprague-Dawley rats were fed in groups of four, for four weeks, protein sufficient and protein deficient diets with or without ethanol. Messenger RNA levels for pancreatic lithostathine were assessed in all four groups. Both ethanol and protein deficiency, separately and in combination, increased mRNA levels for lithostathine. Thus, both chronic ethanol consumption and dietary protein deficiency increase the capacity of the pancreatic acinar cell to synthesize lithostathine.

Alcoholism↗
PubMed2001

Three-dimensional structure of the lithostathine protofibril, a protein involved in Alzheimer's disease.

Neurodegenerative diseases are characterized by the presence of filamentous aggregates of proteins. We previously established that lithostathine is a protein overexpressed in the pre-clinical stages of Alzheimer's disease. Furthermore, it is present in the pathognomonic lesions associated with Alzheimer's disease. After self-proteolysis, the N-terminally truncated form of lithostathine leads to the formation of fibrillar aggregates. Here we observed using atomic force microscopy that these aggregates consisted of a network of protofibrils, each of which had a twisted appearance. Electron microscopy and image analysis showed that this twisted protofibril has a quadruple helical structure. Three-dimensional X-ray structural data and the results of biochemical experiments showed that when forming a protofibril, lithostathine was first assembled via lateral hydrophobic interactions into a tetramer. Each tetramer then linked up with another tetramer as the result of longitudinal electrostatic interactions. All these results were used to build a structural model for the lithostathine protofibril called the quadruple-helical filament (QHF-litho). In conclusion, lithostathine strongly resembles the prion protein in its dramatic proteolysis and amyloid proteins in its ability to form fibrils.

Alzheimer Disease↗
PubMed1992

Evidence that human kidney produces a protein similar to lithostathine, the pancreatic inhibitor of CaCO3 crystal growth.

Pancreatic juice is supersaturated in calcium carbonate. CaCO3 crystal growth is controlled by lithostathine, a secretory protein synthesized by pancreatic acinar cells, first described as a constituent of pancreatic stones. It was recently reported that, in the thin descending limb of the Henle's loop, urine was supersaturated in CaCO3 (Coe FL, Parks JH: Defenses of an unstable compromise: crystallization inhibitors and the kidney's role in mineral regulation. Kidney Int. 1990: 38, 625-631. This observation suggested the presence in kidney of a similar inhibitor. In this study, we show that a protein immunologically related to lithostathine is actually present in urine of healthy subjects and in renal stones. Immunocytochemistry of kidney sections localized the protein to cells of the proximal tubules and thick ascending limbs of the Henle's loops. Protein extracts of renal stones inhibited CaCO3 crystal growth in vitro and this inhibition was significantly lifted by incubating the extracts with antibodies to lithostathine. The protein is not immunologically related to nephrocalcin. Because of its structural and functional similarities with pancreatic lithostathine, it was called renal lithostathine.

Calcium Carbonate↗
PubMed2003

An insight into the role of human pancreatic lithostathine.

Human lithostathine was initially isolated from pancreatic stones in patients with alcoholic calcifying chronic pancreatitis. It is secreted into the pancreatic juice where it was believed to inhibit stone formation. The N-terminal undecapeptide was assumed to play an important role in the mechanism, by adsorption to the crystal surface. Later, the role of lithostathine in calcite formation and growth was questioned, together with the associated mechanism of action. In particular, although lithostathine adsorbs on calcite crystal, this property does not now seem to be specific. Moreover, the N-terminal undecapeptide is not likely to have, by itself, the function of the entire protein. The different aspects of this controversy are reviewed and discussed, particularly in the light of recent structural biology. Comparative biological data now available allow us to draw an interesting parallel between lithostathine and other related proteins. Finally, lithostathine might affect stone formation and may also have another function which could be investigated in the other proteins belonging to the same structural family.

Amino Acid Sequence↗
PubMed2003

Lithostathine quadruple-helical filaments form proteinase K-resistant deposits in Creutzfeldt-Jakob disease.

Autocatalytic cleavage of lithostathine leads to the formation of quadruple-helical fibrils (QHF-litho) that are present in Alzheimer's disease. Here we show that such fibrils also occur in Creutzfeldt-Jakob and Gerstmann-Sträussler-Scheinker diseases, where they form protease-K-resistant deposits and co-localize with amyloid plaques formed from prion protein. Lithostathine does not appear to change its native-like, globular structure during fibril formation. However, we obtained evidence that a cluster of six conserved tryptophans, positioned around a surface loop, could act as a mobile structural element that can be swapped between adjacent protein molecules, thereby enabling the formation of higher order fibril bundles. Despite their association with these clinical amyloid deposits, QHF-litho differ from typical amyloid fibrils in several ways, for example they produce a different infrared spectrum and cannot bind Congo Red, suggesting that they may not represent amyloid structures themselves. Instead, we suggest that lithostathine constitutes a novel component decorating disease-associated amyloid fibrils. Interestingly, [6,6']bibenzothiazolyl-2,2'-diamine, an agent found previously to disrupt aggregates of huntingtin associated with Huntington's disease, can dissociate lithostathine bundles into individual protofilaments. Disrupting QHF-litho fibrils could therefore represent a novel therapeutic strategy to combat clinical amyloidoses.

Amino Acid Sequence↗
PubMed1996

What function for human lithostathine?: structural investigations by three-dimensional structure modeling and high-resolution NMR spectroscopy.

Human lithostathine is a 144-residue protein, expressed in various organs and pathologies. Several biological functions have been proposed for this protein. Among others, inhibition of nucleation and growth of CaCO3 crystals in the pancreas and bacterial aggregation has retained attention, because lithostathine presents high sequence similarities with calcium-dependent (or C-type) lectins. To study its structure-function relationship and compare it with that of C-type lectins, we have built a model for lithostathine. This model is derived from the only two C-type lectins of known structures: rat mannose binding protein and human E-selectin. An original strategy, inspired by that proposed by Havel and Snow, was designed for model building. We have undertaken NMR studies on the natural protein. Although complete structure determination has not yet been achieved, the NMR studies did confirm the main characteristics of the model. From analysis of the proposed model, we concluded that lithostathine is not expected to present sugar- or calcium-binding properties. Therefore, the mechanisms of bacterial aggregation and inhibition of CaCO3 nucleation and growth have not yet been elucidated.

Amino Acid Sequence↗
PubMed1993

[Renal lithostathine: a new protein inhibitor of lithogenesis].

Lithostathine is a protein of pancreatic secretion inhibiting calcium carbonate crystal growth. Antibodies to lithostathine were used to identify a related protein in urine and kidney stones. Western blot analysis of proteins extracted from concentrated normal urine or kidney stones demonstrated the presence of a protein with an apparent molecular weight of 23 kDa. The same antibodies were used in immunolocalization experiments on fresh human nephrectomy specimens cryosections. A positive signal was observed in the cells of proximal tubules and thick ascending limbs of Henle's loop. Protein extracts of renal stones inhibited calcium carbonate crystal growth. Because of its structural and functional similarities with pancreatic lithostathine, it was called renal lithostathine.

Animals↗