RESUMEN
Vacuolar ATPases (V-ATPases), proton pumps composed of 16 subunits, are necessary for a variety of cellular functions. Subunit "a" has four isoforms, a1-a4, each with a distinct cellular location. We identified a phosphoinositide (PIP) interaction motif, KXnK(R)IK(R), conserved in all four isoforms, and hypothesize that a/PIP interactions regulate V-ATPase recruitment/retention to different organelles. Among the four isoforms, a2 is enriched on Golgi with a2 mutations in the PIP motif resulting in cutis laxa. We hypothesize that the hydrophilic N-terminal (NT) domain of a2 contains a lipid-binding domain, and mutations in this domain prevent interaction with Golgi-enriched PIPs, resulting in cutis laxa. We recreated the cutis laxa-causing mutation K237_V238del, and a double mutation in the PIP-binding motif, K237A/V238A. Circular dichroism confirmed that there were no protein structure alterations. Pull-down assays with PIP-enriched liposomes revealed that wildtype a2NT preferentially binds phosphatidylinositol 4-phosphate (PI(4)P), while mutants decreased binding to PI(4)P. In HEK293 cells, wildtype a2NT was localized to Golgi and co-purified with microsomal membranes. Mutants reduced Golgi localization and membrane association. Rapamycin depletion of PI(4)P diminished a2NT-Golgi localization. We conclude that a2NT is sufficient for Golgi retention, suggesting the lipid-binding motif is involved in V-ATPase targeting and/or retention. Mutational analyses suggest a molecular mechanism underlying how a2 mutations result in cutis laxa.
Asunto(s)
Cutis Laxo , ATPasas de Translocación de Protón Vacuolares , Humanos , Cutis Laxo/genética , Cutis Laxo/metabolismo , Células HEK293 , Isoformas de Proteínas/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , MutaciónRESUMEN
Vacuolar ATPases (V-ATPases) are multi-subunit ATP-dependent proton pumps necessary for cellular functions, including pH regulation and membrane fusion. The evidence suggests that the V-ATPase a-subunit's interaction with the membrane signaling lipid phosphatidylinositol (PIPs) regulates the recruitment of V-ATPase complexes to specific membranes. We generated a homology model of the N-terminal domain of the human a4 isoform (a4NT) using Phyre2.0 and propose a lipid binding domain within the distal lobe of the a4NT. We identified a basic motif, K234IKK237, critical for interaction with phosphoinositides (PIP), and found similar basic residue motifs in all four mammalian and both yeast a-isoforms. We tested PIP binding of wildtype and mutant a4NT in vitro. In protein lipid overlay assays, the double mutation K234A/K237A and the autosomal recessive distal renal tubular-causing mutation K237del reduced both PIP binding and association with liposomes enriched with PI(4,5)P2, a PIP enriched within plasma membranes. Circular dichroism spectra of the mutant protein were comparable to wildtype, indicating that mutations affected lipid binding, not protein structure. When expressed in HEK293, wildtype a4NT localized to the plasma membrane in fluorescence microscopy and co-purified with the microsomal membrane fraction in cellular fractionation experiments. a4NT mutants showed reduced membrane association and decreased plasma membrane localization. Depletion of PI(4,5)P2 by ionomycin caused reduced membrane association of the WT a4NT protein. Our data suggest that information contained within the soluble a4NT is sufficient for membrane association and that PI(4,5)P2 binding capacity is involved in a4 V-ATPase plasma membrane retention.
Asunto(s)
ATPasas de Translocación de Protón Vacuolares , Animales , Humanos , Células HEK293 , ATPasas de Translocación de Protón Vacuolares/metabolismo , Saccharomyces cerevisiae/metabolismo , Isoformas de Proteínas/metabolismo , Membrana Celular/metabolismo , Fosfatidilinositoles/metabolismo , Sitios de Unión , Mamíferos/metabolismoRESUMEN
Vacuolar H+-ATPases (V-ATPases) are large, multisubunit proton pumps that acidify the lumen of organelles in virtually every eukaryotic cell and in specialized acid-secreting animal cells, the enzyme pumps protons into the extracellular space. In higher organisms, most of the subunits are expressed as multiple isoforms, with some enriched in specific compartments or tissues and others expressed ubiquitously. In mammals, subunit a is expressed as four isoforms (a1-4) that target the enzyme to distinct biological membranes. Mutations in a isoforms are known to give rise to tissue-specific disease, and some a isoforms are upregulated and mislocalized to the plasma membrane in invasive cancers. However, isoform complexity and low abundance greatly complicate purification of active human V-ATPase, a prerequisite for developing isoform-specific therapeutics. Here, we report the purification of an active human V-ATPase in native lipid nanodiscs from a cell line stably expressing affinity-tagged a isoform 4 (a4). We find that exogenous expression of this single subunit in HEK293F cells permits assembly of a functional V-ATPase by incorporation of endogenous subunits. The ATPase activity of the preparation is >95% sensitive to concanamycin A, indicating that the lipid nanodisc-reconstituted enzyme is functionally coupled. Moreover, this strategy permits purification of the enzyme's isolated membrane subcomplex together with biosynthetic assembly factors coiled-coil domain-containing protein 115, transmembrane protein 199, and vacuolar H+-ATPase assembly integral membrane protein 21. Our work thus lays the groundwork for biochemical characterization of active human V-ATPase in an a subunit isoform-specific manner and establishes a platform for the study of the assembly and regulation of the human holoenzyme.
Asunto(s)
ATPasas de Translocación de Protón Vacuolares , Transporte Biológico , Membrana Celular/metabolismo , Humanos , Saccharomyces cerevisiae/metabolismoRESUMEN
Autosomal recessive osteopetrosis (ARO) is a severe genetic bone disease characterized by high bone density due to mutations that affect formation or function of osteoclasts. Mutations in the a3 subunit of the vacuolar-type H+ -ATPase (encoded by T-cell immune regulator 1 [TCIRG1]) are responsible for ~50% of all ARO cases. We identified a novel TCIRG1 (c.G630A) mutation responsible for an unusually mild form of the disease. To characterize this mutation, osteoclasts were differentiated using peripheral blood monocytes from the patient (c.G630A/c.G630A), male sibling (+/+), unaffected female sibling (+/c.G630A), and unaffected parent (+/c.G630A). Osteoclast formation, bone-resorbing function, TCIRG1 protein, and mRNA expression levels were assessed. The c.G630A mutation did not affect osteoclast differentiation; however, bone-resorbing function was decreased. Both TCIRG1 protein and full-length TCIRG1 mRNA expression levels were also diminished in the affected patient's sample. The c.G630A mutation replaces the last nucleotide of exon 6 and may cause splicing defects. We analyzed the TCIRG1 splicing pattern between exons 4 to 8 and detected deletions of exons 5, 6, 7, and 5-6 (ΔE56). These deletions were only observed in c.G630A/c.G630A and +/c.G630A samples, but not in +/+ controls. Among these deletions, only ΔE56 maintained the reading frame and was predicted to generate an 85 kDa protein. Exons 5-6 encode an uncharacterized portion of the cytoplasmic N-terminal domain of a3, a domain not involved in proton translocation. To investigate the effect of ΔE56 on V-ATPase function, we transformed yeast with plasmids carrying full-length or truncated Vph1p, the yeast ortholog of a3. Both proteins were expressed; however, ΔE56-Vph1p transformed yeast failed to grow on Zn2+ -containing plates, a growth assay dependent on V-ATPase-mediated vacuolar acidification. In conclusion, our results show that the ΔE56 truncated protein is not functional, suggesting that the mild ARO phenotype observed in the patient is likely due to the residual full-length protein expression.
Asunto(s)
Empalme Alternativo , Huesos/metabolismo , Osteoclastos/metabolismo , Osteopetrosis/genética , Mutación Puntual , Eliminación de Secuencia , ATPasas de Translocación de Protón Vacuolares/genética , Adolescente , Densidad Ósea , Huesos/diagnóstico por imagen , Huesos/patología , Niño , Trastornos de los Cromosomas , Exones , Genes Recesivos , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Madres , Osteoclastos/patología , Osteopetrosis/diagnóstico por imagen , Osteopetrosis/metabolismo , Osteopetrosis/patología , Cultivo Primario de Células , Estructura Secundaria de Proteína , Hermanos , Tomografía Computarizada por Rayos X , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/deficienciaRESUMEN
The a subunit is the largest of 15 different subunits that make up the vacuolar H+-ATPase (V-ATPase) complex, where it functions in proton translocation. In mammals, this subunit has four paralogous isoforms, a1-a4, which may encode signals for targeting assembled V-ATPases to specific intracellular locations. Despite the functional importance of the a subunit, its structure remains controversial. By studying molecular mechanisms of human disease-causing missense mutations within a subunit isoforms, we may identify domains critical for V-ATPase targeting, activity and/or regulation. cDNA-encoded FLAG-tagged human wildtype ATP6V0A2 (a2) and ATP6V0A4 (a4) subunits and their mutants, a2P405L (causing cutis laxa), and a4R449H and a4G820R (causing renal tubular acidosis, dRTA), were transiently expressed in HEK 293 cells. N-Glycosylation was assessed using endoglycosidases, revealing that a2P405L, a4R449H, and a4G820R were fully N-glycosylated. Cycloheximide (CHX) chase assays revealed that a2P405L and a4R449H were unstable relative to wildtype. a4R449H was degraded predominantly in the proteasomal pathway, whereas a2P405L was degraded in both proteasomal and lysosomal pathways. Immunofluorescence studies disclosed retention in the endoplasmic reticulum and defective cell-surface expression of a4R449H and defective Golgi trafficking of a2P405L Co-immunoprecipitation studies revealed an increase in association of a4R449H with the V0 assembly factor VMA21, and a reduced association with the V1 sector subunit, ATP6V1B1 (B1). For a4G820R, where stability, degradation, and trafficking were relatively unaffected, 3D molecular modeling suggested that the mutation causes dRTA by blocking the proton pathway. This study provides critical information that may assist rational drug design to manage dRTA and cutis laxa.
Asunto(s)
Acidosis Tubular Renal/genética , Cutis Laxo/genética , Modelos Moleculares , Mutación Missense , Procesamiento Proteico-Postraduccional , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón Vacuolares/genética , Acidosis Tubular Renal/metabolismo , Acidosis Tubular Renal/patología , Sustitución de Aminoácidos , Membrana Celular/enzimología , Membrana Celular/metabolismo , Membrana Celular/patología , Cutis Laxo/metabolismo , Cutis Laxo/patología , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Estabilidad de Enzimas , Glicosilación , Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Aparato de Golgi/patología , Células HEK293 , Humanos , Riñón/enzimología , Riñón/metabolismo , Riñón/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Transporte de Proteínas , Proteolisis , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
The a subunit of the V0 membrane-integrated sector of human V-ATPase has four isoforms, a1-a4, with diverse and crucial functions in health and disease. They are encoded by four conserved paralogous genes, and their vertebrate orthologs have positionally conserved N-glycosylation sequons within the second extracellular loop, EL2, of the a subunit membrane domain. Previously, we have shown directly that the predicted sequon for the a4 isoform is indeed N-glycosylated. Here we extend our investigation to the other isoforms by transiently transfecting HEK 293 cells to express cDNA constructs of epitope-tagged human a1-a3 subunits, with or without mutations that convert Asn to Gln at putative N-glycosylation sites. Expression and N-glycosylation were characterized by immunoblotting and mobility shifts after enzymatic deglycosylation, and intracellular localization was determined using immunofluorescence microscopy. All unglycosylated mutants, where predicted N-glycosylation sites had been eliminated by sequon mutagenesis, showed increased relative mobility on immunoblots, identical to what was seen for wild-type a subunits after enzymatic deglycosylation. Cycloheximide-chase experiments showed that unglycosylated subunits were turned over at a higher rate than N-glycosylated forms by degradation in the proteasomal pathway. Immunofluorescence colocalization analysis showed that unglycosylated a subunits were retained in the ER, and co-immunoprecipitation studies showed that they were unable to associate with the V-ATPase assembly chaperone, VMA21. Taken together with our previous a4 subunit studies, these observations show that N-glycosylation is crucial in all four human V-ATPase a subunit isoforms for protein stability and ultimately for functional incorporation into V-ATPase complexes.
Asunto(s)
ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Secuencia de Aminoácidos , Asparagina/genética , Sitios de Unión , Retículo Endoplásmico/metabolismo , Glutamina/genética , Glicosilación/efectos de los fármacos , Células HEK293 , Humanos , Mutación , Unión Proteica , Biosíntesis de Proteínas , Estabilidad Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
The a subunit is the largest of 14 different subunits that make up the V-ATPase complex. In mammalian species this membrane protein has four paralogous isoforms, a1-a4. Clinically, a subunit isoforms are implicated in diverse diseases; however, little is known about their structure and function. The subunit has conserved, predicted N-glycosylation sites, and the a3 isoform has been directly shown to be N-glycosylated. Here we ask if human a4 (ATP6V0A4) is N-glycosylated at the predicted site, Asn489. We transfected HEK 293 cells, using the pCDNA3.1 expression-vector system, to express cDNA constructs of epitope-tagged human a4 subunit, with or without mutations to eliminate the putative glycosylation site. Glycosylation was characterized also by treatment with endoglycosidases; expression and localization were assessed by immunoblotting and immunofluorescence. Endoglycosidase-treated wild type (WT) a4 showed increased relative mobility on immunoblots, compared with untreated WT a4. This relative mobility was identical to that of unglycosylated mutant a4N489D , demonstrating that the a4 subunit is glycosylated. Cycloheximide pulse-chase experiments showed that the unglycosylated subunit degraded at a higher rate than the N-glycosylated form. Unglycosylated a4 was degraded mostly in the proteasomal pathway, but also, in part, through the lysosomal pathway. Immunofluorescence colocalization data showed that unglycosylated a4 was mostly retained in the ER, and that plasma membrane trafficking was defective. Co-immunoprecipitation studies suggested that a4N489D does not assemble with the V-ATPase V1 domain. Taken together, these data show that N-glycosylation plays a crucial role in a4 stability, and in V-ATPase assembly and trafficking to the plasma membrane. J. Cell. Biochem. 117: 2757-2768, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Membrana Celular/metabolismo , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/metabolismo , Secuencia de Aminoácidos , Western Blotting , Técnica del Anticuerpo Fluorescente , Glicosilación , Células HEK293 , Humanos , Inmunoprecipitación , Estabilidad Proteica , Subunidades de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Published topological models of the integral membrane a subunit of the vacuolar proton-translocating ATPase complex have not been in agreement with respect to either the number of transmembrane helices within the integral membrane domain, or their limits and orientations within the lipid bilayer. In the present work we have constructed a predictive model of the membrane insertion of the yeast a subunit, Vph1p, from a consensus of seven topology prediction algorithms. The model was tested experimentally using epitope tagging, green fluorescent protein fusion, and protease accessibility analysis in purified yeast vacuoles. Results suggest that a consensus prediction of eight transmembrane helices with both the amino-terminus and carboxyl-terminus in the cytoplasm is correct. Characterization of two glycosylation sites within the homologous mouse a subunit membrane domain further corroborates this topology. Moreover, the model takes into account published data on cytoplasmic and luminal accessibility of specific amino acids. Changes in the degree of protease accessibility in response to the V-ATPase substrate, MgATP, and the V-ATPase-specific inhibitor, concanamycin A, suggest that functional conformational changes occur in the large cytoplasmic loop between TM6 and TM7 of Vph1p. These data substantially confirm one topological model of the V-ATPase a subunit and support the notion that conformational changes occur within the membrane domain, possibly involving previously proposed axial rotation and/or linear displacement of TM7 in the proton transport cycle.
Asunto(s)
Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/enzimología , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/metabolismo , Electroforesis en Gel de Poliacrilamida , Metabolismo Energético , Glicosilación , Estructura Terciaria de Proteína , Subunidades de Proteína/químicaRESUMEN
V-ATPase-mediated acid secretion is required for osteoclast bone resorption. Osteoclasts are enriched in V-ATPase a3 and d2 subunit isoforms, and disruption of either of their genes impairs bone resorption. Using purified fusion proteins of a3 N-terminal domain (NTa3) and full-length d subunits we determined in a solid-phase binding assay that half-maximal binding of d1 or d2 to immobilized NTa3 occurs at 3.1 ± 0.4 or 3.6 ± 0.6 nM, respectively, suggesting equally high-affinity interactions. A high-throughput modification of this assay was then used to screen chemical libraries for a3-d2 interaction inhibitors, and luteolin, a naturally occurring flavonoid, was identified, with half-maximal inhibition at 2.4 ± 0.9 µM. Luteolin did not significantly affect NIH/3T3 or RAW 264.7 cell viability, nor did it affect cytokine-induced osteoclastogenesis of RAW 264.7 cells or bone marrow mononuclear cells at concentrations ≤ 40 µM. Luteolin inhibited osteoclast bone resorption with an EC(50) of approximately 2.5 µM, without affecting osteoclast actin ring formation. Luteolin-treated osteoclasts produced deeper resorption pits, but with decreased surface area, resulting in overall decreased pit volume. Luteolin did not affect transcription, or protein levels, of V-ATPase subunits a3, d2, and E, or V(1) V(0) assembly. Previous work has shown that luteolin can be effective in reducing bone resorption, and our studies suggest that this effect of luteolin may be through disruption of osteoclast V-ATPase a3-d2 interaction. We conclude that the V-ATPase a3-d2 interaction is a viable target for novel anti-resorptive therapeutics that potentially preserve osteoclast-osteoblast signaling important for bone remodeling.
Asunto(s)
Resorción Ósea/patología , Luteolina/farmacología , Complejos Multiproteicos/antagonistas & inhibidores , Osteoclastos/efectos de los fármacos , ATPasas de Translocación de Protón Vacuolares/metabolismo , Actinas/metabolismo , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Isoenzimas/metabolismo , Ratones , Células 3T3 NIH , Osteoclastos/metabolismo , Osteoclastos/patología , Osteogénesis , Mapeo de Interacción de Proteínas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Vacuolar H(+)-ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-κB ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC(50) of â¼1.2 µm on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-κB ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis.
Asunto(s)
Resorción Ósea/enzimología , Regulación hacia Abajo , Osteoclastos/enzimología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Resorción Ósea/genética , Línea Celular , Humanos , Ratones , Osteoclastos/química , Unión Proteica , Estructura Terciaria de Proteína , Técnicas del Sistema de Dos Híbridos , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
V-ATPases are multimeric proton pumps. The 100-kDa "a" subunit is encoded by four isoforms (a1-a4) in mammals and two (Vph1p and Stv1p) in yeast. a3 is enriched in osteoclasts and is essential for bone resorption, whereas a4 is expressed in the distal nephron and acidifies urine. Mutations in human a3 and a4 result in osteopetrosis and distal renal tubular acidosis, respectively. Human a3 (G405R and R444L) and a4 (P524L and G820R) mutations were recreated in the yeast ortholog Vph1p, a3 (G424R and R462L), and a4 (W520L and G812R). Mutations in a3 resulted in wild type vacuolar acidification and growth on media containing 4 mM ZnCl2, 200 mM CaCl2, or buffered to pH 7.5 with V-ATPase hydrolytic and pumping activity decreased by 30-35%. Immunoblots confirmed wild type levels for V-ATPase a, A, and B subunits on vacuolar membranes. a4 G812R resulted in defective growth on selective media with V-ATPase hydrolytic and pumping activity decreased by 83-85% yet with wild type levels of a, A, and B subunits on vacuolar membranes. The a4 W520L mutation had defective growth on selective media with no detectable V-ATPase activity and reduced expression of a, A, and B subunits. The a4 W520L mutation phenotypes were dominant negative, as overexpression of wild type yeast a isoforms, Vph1p, or Stv1p, did not restore growth. However, deletion of endoplasmic reticulum assembly factors (Vma12p, Vma21p, and Vma22p) partially restored a and B expression. That a4 W520L affects both Vo and V1 subunits is a unique phenotype for any V-ATPase subunit mutation and supports the concerted pathway for V-ATPase assembly in vivo.
Asunto(s)
Acidosis Tubular Renal , Isoenzimas , Mutación , Osteopetrosis , Subunidades de Proteína , Proteínas de Saccharomyces cerevisiae , ATPasas de Translocación de Protón Vacuolares , Acidosis Tubular Renal/enzimología , Acidosis Tubular Renal/genética , Adenosina Trifosfato/metabolismo , Animales , Inhibidores Enzimáticos/metabolismo , Genotipo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Macrólidos/metabolismo , Ratones , Osteopetrosis/enzimología , Osteopetrosis/genética , Fenotipo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Vacuolas/químicaRESUMEN
Osteoclasts dissolve bone through acidification of an extracellular compartment by means of a multimeric vacuolar type H+-ATPase (V-ATPase). In mammals, there are four isoforms of the 100-kDa V-ATPase "a" subunit. Mutations in the a3 isoform result in deficient bone resorption and osteopetrosis, suggesting that a3 has a unique function in osteoclasts. It is thus surprising that several studies show a basal level of a3 expression in most tissues. To address this issue, we have compared a3 expression in bone with expression in other tissues. RNA blots revealed that the a3 isoform was expressed highest in bone and confirmed its expression (in decreasing order) in liver, kidney, brain, lung, spleen, and muscle. In situ hybridization on bone tissue sections revealed that the a3 isoform was highly expressed in multinucleated osteoclasts but not in mononuclear stromal cells, whereas the a1 isoform was expressed in both cell types at about the same level. We also found that a3 expression was greater in osteoclasts with 10 or more nuclei as compared with osteoclasts with five or fewer nuclei. We hypothesize that these differences in a3 expression may be associated with previously demonstrated differences between large and small osteoclasts with reference to their resorptive activity.
Asunto(s)
Isoenzimas/metabolismo , Osteoclastos/enzimología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Concentración de Iones de Hidrógeno , Hibridación in Situ , Isoenzimas/química , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ATPasas de Translocación de Protón Vacuolares/químicaRESUMEN
Aggrecan is the major proteoglycan in the extracellular matrix of cartilage. A notable exception is nanomelic cartilage, which lacks aggrecan in its matrix. The example of nanomelia and other evidence leads us to believe that the G3 domain plays an important role in aggrecan processing, and it has indeed been confirmed that G3 allows glycosaminoglycan (GAG) chain attachment and product secretion. However, it is not clear how G3, which contains at least a carbohydrate recognition domain (CRD) and a complement binding protein (CBP) motif, plays these two functional roles. The present study was designed to dissect the mechanisms of this phenomenon and specially 1) to determine the effects of various cysteine residues in GAG modification and product secretion as well as 2) to investigate which of the two processing events is the critical step in the product processing. Our studies demonstrated that removal of the two amino-terminal cysteines in the CRD motif and the single cysteine in the amino terminus of CBP inhibited secretion of CRD and CBP. Use of the double mutant CRD construct also allowed us to observe a deviation from the usual strict coupling of GAG modification and product secretion steps. The presence of a small chondroitin sulfate fragment overcame the secretion-inhibitory effects once the small chondroitin sulfate fragment was modified by GAG.