RESUMEN
Bone biomineralization is mediated by a special class of extracellular vesicles, named matrix vesicles (MVs), released by osteogenic cells. The MV membrane is enriched in sphingomyelin (SM), cholesterol (Chol) and tissue non-specific alkaline phosphatase (TNAP) compared with the parent cells' plasma membrane. TNAP is an ATP phosphohydrolase bound to cell and MV membranes via a glycosylphosphatidylinositol (GPI) anchor. Previous studies have shown that the lipid microenvironment influences the catalytic activity of enzymes incorporated into lipid bilayers. However, there is a lack of information about how the lipid microenvironment controls the ability of MV membrane-bound enzymes to induce mineral precipitation. Herein, we used TNAP-harboring proteoliposomes made of either pure dimyristoylphosphatidylcholine (DMPC) or DMPC mixed with either Chol, SM or both of them as MV biomimetic systems to evaluate how the composition modulates the lipid microenvironment and, in turn, TNAP incorporation into the lipid bilayer by means of calorimetry. These results were correlated with the proteoliposomes' catalytic activity and ability to induce the precipitation of amorphous calcium phosphate (ACP) in vitro. DMPC:SM proteoliposomes displayed the highest efficiency of mineral propagation, apparent affinity for ATP and substrate hydrolysis efficiency, which correlated with their highest degree of membrane organization (highest ΔH), among the tested proteoliposomes. Results obtained from turbidimetry and Fourier transformed infrared (FTIR) spectroscopy showed that the tested proteoliposomes induced ACP precipitation with the order DMPC:SM>DMPC:Chol:SM≈DMPC:Chol>DMPC which correlated with the lipid organization and the presence of SM in the proteoliposome membrane. Our study arises important insights regarding the physical properties and role of lipid organization in MV-mediated mineralization.
Asunto(s)
Adenosina Trifosfato/metabolismo , Fosfatasa Alcalina/metabolismo , Biomineralización/fisiología , Fosfatos de Calcio/metabolismo , Liposomas/metabolismo , Proteolípidos/metabolismo , Animales , Bovinos , Colesterol/química , Dimiristoilfosfatidilcolina/química , Hidrólisis , Liposomas/química , Proteolípidos/química , Ratas , Esfingomielinas/químicaRESUMEN
Previous studies have demonstrated a negative correlation between intestinal alkaline phosphatase (IAP) activity and calcium (Ca) absorption in the gut, as IAP acts as a protective mechanism inhibiting high Ca entry into enterocytes, preventing Ca overload. Here we evaluated Ca absorption and bone properties in knockout mice (KO) completely devoid of duodenal IAP (Akp3 -/- mice). Female C57BL/6 control mice (WT, n = 7) and KO mice (n = 10) were used to determine Ca absorption in vivo and by in situ isolated duodenal loops followed by histomorphometric analysis of duodenal villi and crypts. Bone mineral density, morphometry, histomorphometry and trabecular connectivity and biomechanical properties were measured on bones. We observed mild atrophy of the villi with lower absorption surface and a significantly higher Ca uptake in KO mice. While no changes were seen in cortical bone, we found better trabecular connectivity and biomechanical properties in the femurs of KO mice compared to WT mice. Our data indicate that IAP KO mice display higher intestinal Ca uptake, which over time appears to correlate with a positive effect on the biomechanical properties of trabecular bone.
Asunto(s)
Fosfatasa Alcalina/deficiencia , Calcio/metabolismo , Hueso Esponjoso/metabolismo , Intestinos/enzimología , Fosfatasa Alcalina/metabolismo , Animales , Fenómenos Biomecánicos , Densidad Ósea , Calcio/sangre , Duodeno/metabolismo , Femenino , Fracturas del Cuello Femoral/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatos/sangreRESUMEN
Mineralization of the skeleton starts within cell-derived matrix vesicles (MVs); then, minerals propagate to the extracellular collagenous matrix. Tissue-nonspecific alkaline phosphatase (TNAP) degrades inorganic pyrophosphate (PPi), a potent inhibitor of mineralization, and contributes Pi (Phosphate) from ATP to initiate mineralization. Compared to the plasma membrane, MVs are rich in Cholesterol (Chol) (â¼32%) and TNAP, but how Chol influences TNAP activity remains unclear. We have reconstituted TNAP in liposomes of dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) combined with Chol or its derivatives Cholestenone (Achol) and Ergosterol (Ergo). DPPC plus 36% sterols in liposome increased the catalytic activity of TNAP toward ATP. The presence of Chol also increased the propagation of minerals by 3.4-fold. The catalytic efficiency of TNAP toward ATP was fourfold lower in DOPC proteoliposomes as compared to DPPC proteoliposomes. DOPC proteoliposomes also increased biomineralization by 2.8-fold as compared to DPPC proteoliposomes. TNAP catalyzed the hydrolysis of ATP more efficiently in the case of the proteoliposome consisting of DOPC with 36% Chol. The same behavior emerged with Achol and Ergo. The organization of the lipid and the structure of the sterol influenced the surface tension (γ), the TNAP phosphohydrolytic activity in the monolayer, and the TNAP catalytic efficiency in the bilayers. Membranes in the Lα phase (Achol) provided better kinetic parameters as compared to membranes in the Lo phase (Chol and Ergo). In conclusion, the physical properties and the lateral organization of lipids in proteoliposomes are crucial to control mineral propagation mediated by TNAP activity during mineralization.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Calcificación Fisiológica , Microambiente Celular , Colesterol/química , Minerales/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Colestenonas/química , Colestenonas/metabolismo , Colesterol/metabolismo , Difosfatos/química , Difosfatos/metabolismo , Ergosterol/química , Ergosterol/metabolismo , Liposomas/química , Liposomas/metabolismo , Masculino , Osteoblastos/citología , Osteoblastos/metabolismo , Fosfatos/química , Fosfatos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Ratas Wistar , Propiedades de SuperficieRESUMEN
Tissue-nonspecific alkaline phosphatase (TNAP) plays a crucial role during skeletal mineralization, and TNAP deficiency leads to the soft bone disease hypophosphatasia. TNAP is anchored to the external surface of the plasma membranes by means of a GPI (glycosylphosphatidylinositol) anchor. Membrane-anchored and solubilized TNAP displays different kinetic properties against physiological substrates, indicating that membrane anchoring influences the enzyme function. Here, we used Electron Spin Resonance (ESR) measurements along with spin labeled phospholipids to probe the possible dynamic changes prompted by the interaction of GPI-anchored TNAP with model membranes. The goal was to systematically analyze the ESR data in terms of line shape changes and of alterations in parameters such as rotational diffusion rates and order parameters obtained from non-linear least-squares simulations of the ESR spectra of probes incorporated into DPPC liposomes and proteoliposomes. Overall, the presence of TNAP increased the dynamics and decreased the ordering in the three distinct regions probed by the spin labeled lipids DOPTC (headgroup), and 5- and 16-PCSL (acyl chains). The largest change was observed for 16-PCSL, thus suggesting that GPI-anchored TNAP can give rise to long reaching modifications that could influence membrane processes halfway through the bilayer.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/metabolismo , Fosfatasa Alcalina/metabolismo , Liposomas/metabolismo , Animales , Células CHO , Cricetulus , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Marcadores de SpinRESUMEN
Proteoliposomes are systems that mimic lipid membranes (liposomes) to which a protein has been incorporated or inserted. During the last decade, these systems have gained prominence as tools for biophysical studies on lipid-protein interactions as well as for their biotechnological applications. Proteoliposomes have a major advantage when compared with natural membrane systems, since they can be obtained with a smaller number of lipidic (and protein) components, facilitating the design and interpretation of certain experiments. However, they have the disadvantage of requiring methodological standardization for incorporation of each specific protein, and the need to verify that the reconstitution procedure has yielded the correct orientation of the protein in the proteoliposome system with recovery of its functional activity. In this review, we chose two proteins under study in our laboratory to exemplify the steps necessary for the standardization of the reconstitution of membrane proteins in liposome systems: (1) alkaline phosphatase, a protein with a glycosylphosphatidylinositol anchor, and (2) Na,K-ATPase, an integral membrane protein. In these examples, we focus on the production of the specific proteoliposomes, as well as on their biochemical and biophysical characterization, with emphasis on studies of lipid-protein interactions. We conclude the chapter by highlighting current prospects of this technology for biotechnological applications, including the construction of nanosensors and of a multi-protein nanovesicular biomimetic to study the processes of initiation of skeletal mineralization.
RESUMEN
Tissue-nonspecific alkaline phosphatase (TNAP) is associated to the plasma membrane via a GPI-anchor and plays a key role in the biomineralization process. In plasma membranes, most GPI-anchored proteins are associated with "lipid rafts", ordered microdomains enriched in sphingolipids, glycosphingolipids and cholesterol. In order to better understand the role of lipids present in rafts and their interactions with GPI-anchored proteins, the insertion of TNAP into different lipid raft models was studied using dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), sphingomyelin (SM) and ganglioside (GM1). Thus, the membrane models studied were binary systems (9:1 molar ratio) containing DPPC:Chol, DPPC:SM and DPPC:GM1, ternary systems (8:1:1 molar ratio) containing DPPC:Chol:SM, DPPC:Chol:GM1 and DPPC:SM:GM1 and finally, a quaternary system (7:1:1:1 molar ratio) containing DPPC:Chol:SM:GM1. Calorimetry analysis of the liposomes and proteoliposomes indicate that lateral phase segregation could be noted only in the presence of cholesterol, with the formation of cholesterol-rich microdomains centered above Tc=41.5°C. The presence of GM1 and SM into DPPC-liposomes influenced mainly ΔH and Δt(1/2) values. The gradual increase in the complexity of the systems decreased the activity of the enzyme incorporated. The presence of the enzyme also fluidifies the systems, as seen by the intense reduction in ∆H values, but do not alter Tc values significantly. Therefore, the study of different microdomains and its biophysical characterization may contribute to the knowledge of the interactions between the lipids present in MVs and its interactions with TNAP.
Asunto(s)
Fosfatasa Alcalina/análisis , Liposomas/química , Microdominios de Membrana/química , Proteolípidos/química , 1,2-Dipalmitoilfosfatidilcolina/química , Animales , Células Cultivadas , Colesterol/química , Gangliósido G(M1)/química , Transición de Fase , Ratas , Esfingomielinas/química , TermodinámicaRESUMEN
Tissue-nonspecific alkaline phosphatase (TNAP), present on the surface of chondrocyte- and osteoblast-derived matrix vesicles (MVs), plays key enzymatic functions during endochondral ossification. Many studies have shown that MVs are enriched in TNAP and also in cholesterol compared to the plasma membrane. Here we have studied the influence of cholesterol on the reconstitution of TNAP into dipalmitoylphosphatidylcholine (DPPC)-liposomes, monitoring the changes in lipid critical transition temperature (T(c)) and enthalpy variation (∆H) using differential scanning calorimetry (DSC). DPPC-liposomes revealed a T(c) of 41.5 °C and ∆H of 7.63 Kcal mol(-1). The gradual increase in cholesterol concentration decrease ∆H values, reaching a ∆H of 0.87Kcalmol(-1) for DPPC:cholesterol system with 36mol% of cholesterol. An increase in T(c), up to 47 °C for the DPPC:cholesterol liposomes (36 mol% of Chol), resulted from the increase in the area per molecule in the gel phase. TNAP (0.02 mg/mL) reconstitution was done with protein:lipid 1:10,000 (molar ratio), resulting in 85% of the added enzyme being incorporated. The presence of cholesterol reduced the incorporation of TNAP to 42% of the added enzyme when a lipid composition of 36 mol% of Chol was used. Furthermore, the presence of TNAP in proteoliposomes resulted in a reduction in ∆H. The gradual proportional increase of cholesterol in liposomes results in broadening of the phase transition peak and eventually eliminates the cooperative gel-to-liquid-crystalline phase transition of phospholipids bilayers. Thus, the formation of microdomains may facilitate the clustering of enzymes and transporters known to be functional in MVs during endochondral ossification.
Asunto(s)
Fosfatasa Alcalina/química , Colesterol/química , Liposomas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Fosfatasa Alcalina/metabolismo , Animales , Rastreo Diferencial de Calorimetría , Ratas , TermodinámicaRESUMEN
During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite (HA) seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Ion transporters control the availability of phosphate and calcium needed for HA deposition. The lipidic microenvironment in which MV-associated enzymes and transporters function plays a crucial physiological role and must be taken into account when attempting to elucidate their interplay during the initiation of biomineralization. In this short mini-review, we discuss the potential use of proteoliposome systems as chondrocyte- and osteoblast-derived MVs biomimetics, as a means of reconstituting a phospholipid microenvironment in a manner that recapitulates the native functional MV microenvironment. Such a system can be used to elucidate the interplay of MV enzymes during catalysis of biomineralization substrates and in modulating in vitro calcification. As such, the enzymatic defects associated with disease-causing mutations in MV enzymes could be studied in an artificial vesicular environment that better mimics their in vivo biological milieu. These artificial systems could also be used for the screening of small molecule compounds able to modulate the activity of MV enzymes for potential therapeutic uses. Such a nanovesicular system could also prove useful for the repair/treatment of craniofacial and other skeletal defects and to facilitate the mineralization of titanium-based tooth implants.
Asunto(s)
Huesos/fisiología , Calcificación Fisiológica/fisiología , Lípidos/fisiología , Proteolípidos/fisiología , Animales , Biomimética , Matriz Ósea/fisiología , Huesos/metabolismo , Humanos , Fosfolípidos/fisiologíaRESUMEN
During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite (HA) seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Ion transporters control the availability of phosphate and calcium needed for HA deposition. The lipidic microenvironment in which MV-associated enzymes and transporters function plays a crucial physiological role and must be taken into account when attempting to elucidate their interplay during the initiation of biomineralization. In this short mini-review, we discuss the potential use of proteoliposome systems as chondrocyte- and osteoblast-derived MVs biomimetics, as a means of reconstituting a phospholipid microenvironment in a manner that recapitulates the native functional MV microenvironment. Such a system can be used to elucidate the interplay of MV enzymes during catalysis of biomineralization substrates and in modulating in vitro calcification. As such, the enzymatic defects associated with disease-causing mutations in MV enzymes could be studied in an artificial vesicular environment that better mimics their in vivo biological milieu. These artificial systems could also be used for the screening of small molecule compounds able to modulate the activity of MV enzymes for potential therapeutic uses. Such a nanovesicular system could also prove useful for the repair/treatment of craniofacial and other skeletal defects and to facilitate the mineralization of titanium-based tooth implants.
Asunto(s)
Animales , Humanos , Huesos/fisiología , Calcificación Fisiológica/fisiología , Lípidos/fisiología , Proteolípidos/fisiología , Biomimética , Matriz Ósea/fisiología , Huesos/metabolismo , Fosfolípidos/fisiologíaRESUMEN
Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 +/- 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Matriz Ósea/metabolismo , Vesículas Citoplasmáticas/fisiología , Diáfisis/enzimología , Osificación Heterotópica/enzimología , Animales , Condrocitos/ultraestructura , Diáfisis/ultraestructura , Femenino , Masculino , Microscopía Electrónica de Transmisión , Osificación Heterotópica/patología , Ratas , Ratas WistarRESUMEN
Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.
Asunto(s)
Animales , Femenino , Masculino , Ratas , Fosfatasa Alcalina/metabolismo , Matriz Ósea/metabolismo , Vesículas Citoplasmáticas/fisiología , Diáfisis/enzimología , Osificación Heterotópica/enzimología , Condrocitos/ultraestructura , Diáfisis/ultraestructura , Microscopía Electrónica de Transmisión , Osificación Heterotópica/patología , Ratas WistarRESUMEN
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder that can be caused by mutations in at least three different genes. Several mutations have been identified in PKD1 and PKD2 genes. Most of the mutations found in PKD2 gene are predicted to cause premature termination of the protein. METHODS: We analysed an Argentinian family characterized previously as PKD2. The PKD2 gene was amplified from genomic DNA using 17 primer pairs and the products were analysed by heteroduplex analysis. PCR products that showed a variation by heteroduplex analysis were sequenced directly. The mutation was confirmed by sequencing relatives. The segregation of the mutation in this family was verified by restriction endonuclease digestion of PCR products obtained from genomic DNA of all family members. Results and conclusions. Here, we report a novel mutation present in an Argentinian family characterized as PKD2 by linkage analysis. The mutation, shared by all affected members of the family, is a thymidine insertion at position 2436 of the gene, which results in a translation frameshift and creates an immediate stop codon. This mutation is expected to lead to a truncated protein that lacks the interacting domain with the PKD1 gene product. The thymidine insertion abolished a Ddel restriction site, allowing a rapid test for detection of PKD2 carriers in the family.
Asunto(s)
Canales de Calcio/genética , Codón de Terminación/genética , Mutación del Sistema de Lectura , Proteínas de la Membrana/genética , Riñón Poliquístico Autosómico Dominante/genética , Adulto , Anciano , Anciano de 80 o más Años , ADN/análisis , Cartilla de ADN/química , Exones , Ligamiento Genético/genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Linaje , Riñón Poliquístico Autosómico Dominante/metabolismo , Reacción en Cadena de la Polimerasa , Pronóstico , Canales Catiónicos TRPPRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disorder with genetic heterogeneity. Up to three loci are involved in this disease, PKD1 on chromosome 16p13.3, PKD2 on 4q21, and a third locus of unknown location. Here we report the existence of locus heterogeneity for this disease in the Argentinian population by performing linkage analysis on 12 families of Caucasian origin. Eleven families showed linkage to PKD 1 and one family showed linkage to PKD2. Two recombinants in the latter family placed the locus PKD2 proximal to D4S1563, in agreement with data recently published on the cloning of this gene. Analysis of clinical data suggests a milder ADPKD phenotype for the PKD2 family.
Asunto(s)
Heterogeneidad Genética , Proteínas de la Membrana/genética , Riñón Poliquístico Autosómico Dominante/genética , Proteínas/genética , Adolescente , Adulto , Argentina , Niño , Preescolar , Femenino , Ligamiento Genético , Humanos , Lactante , Masculino , Linaje , Canales Catiónicos TRPP , Población Blanca/genéticaRESUMEN
OBJECTIVE: To identify the defining characteristics of Primary (PC) and Specialist Care (SC), along with the level of concordance between managers in both sectors in the definition of these characteristics. SETTING: Intermediate course in Administration of the Andalucian School of Public Health, Granada. DESIGN: By means of the Philips 66 technique, followed by consensus construction, the enumeration of the characteristics of care sectors was requested of the managers, who had been previously selected for an Administration course. The procedure was repeated on five different occasions between 1991 and 1993. For each technique four groups were created (two formed by PC and two by SC professionals), in order to obtain self-referred (PC professionals assess PC and the SC ones, SC) and crossed assessments (PC professionals assess SC and vice versa). PARTICIPANTS: 116 professionals in all took part, 55 from the PC sphere, 45 SC and 16 in provincial and/or central services. RESULTS: There was dissonance in the expectations that managers placed in each sector of the health care system. PC managers' views of SC and SC managers' of PC did not coincide with the views of each group of managers on their own health care sector. CONCLUSIONS: When designing activities to coordinate between sectors, a period of clarification, both on the characteristics of each sector and on the expectations placed on each one, should be included. The subsequent negotiation of these can assist the development and maintenance of joint activities to improve coordination.